[41]
E S R OF RETINOIDS IN PI-IOSPHOLIPID MEMBRANES
383
[41] I n t e r a c t i o n s o f R e t i n o i d s w i t h P h o s p h o l i p i d M e m b r a n e s : Electron Spin Resonance
By STEPHEN R. WASSALL and WILLIAM STILLWELL Introduction Retinoids are essential for the maintenance of health. ~Their amphiphilic nature suggests that they will locate within the phospholipid bilayer of membranes, which may constitute a site of action. In addition, the toxicity of high doses of retinoids poses a serious problem to their claimed efficacy in treating a number of cancers. 2 Membrane disruption may be a cause. Several studies have demonstrated that retinoids affect the properties of membranes. Enhanced permeability to nonelectrolytes and ions on incorporation of retinoids into phospholipid membranes has been extensively reported. 3-5 Calorimetric and optical methods have shown that alltrans-retinol, retinal, and retinoic acid broaden the gel to liquid crystalline phase transition and depress its onset temperature in PC (phosphatidylcholine) bilayers. 4,6 Reduced microviscosities in rat erythrocytes and mouse fibroblasts caused by retinoids have been measured by fluorescence polarization, 7,8 whereas magnetic resonance techniques have detected retinoid-associated changes in molecular ordering and dynamics within phospholipid model membranes. 9-13 Further work, however, is required to establish a clear picture. In this chapter, the application of ESR (electron spin resonance) spin label techniques as a method of assay for the interactions of retinoids with 1 L. M. DeLuca and S. S. Shapiro (eds.), Ann. N.Y. Acad. Sci. 359 (1981). 2 D. F. Birt, Proc. Soc. Exp. Biol. Med. 183, 311 (1986). 3 W. Stillwell and M. Ricketts, Biochem. Biophys. Res. Comrnun. 97, 148 (1980). 4 W. Stillwell, M. Ricketts, H. Hudson, and S. Nahmias, Biochim. Biophys. Acta 688, 653 (1982). 5 W. Stillwell and L. Bryant, Biochim. Biophys. Acta 731, 483 (1983). 6 S. R. Wassail, W. Stillwell, and M. Zeldin, Proc. Indiana Acad. Sci. 95, 149 (1986). 7 R. G. Meeks, D. Zaharevitz, and R. F. Chen, Arch. Biochem. Biophys. 207, 141 (1981). 8 A. M. Jetten, R. G. Meeks, and L. M. DeLuca, Ann. N.Y. Acad. Sci. 359, 398 (1981). 9 S. P. Verma, H. Schneider, and I. C. P. Smith, Arch. Biochem. Biophys. 162, 48 (1974). l0 S. R. Wassail and W. Stiilwell, Bull. Magn. Reson. 9, 85 (1987). N C. A. Langsford, M. R. Albrecht, T. M. Phelps, W. Stillwell, and S. R. Wassail, Biophys. J. 51, 239a (1987). 12 S. R. Wassall, T. M. Phelps, M. R. Albrecht, C. A. Langsford, and W. Stillwell, Biochim. Biophys. Acta 939, 393 (1988). i3 H. DeBoeck and R. Zidovetzki, Biochim. Biophys. Acta 946, 244 (1988).
METHODS IN ENZYMOLOGY, VOL. 189
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
384
RECEPTORS, TRANSPORT, AND BINDING PROTEINS
[41]
phospholipid membranes is reviewed. The utility of the approach in membrane research is well documented. 14This chapter focuses on recent studies of the effects of retinoids on order and fluidity within phospholipid model membranes, which demonstrated a distinction between the effects of all-trans-retinoic acid and those of all-trans-retinol and retinal. 10-12Preliminary ESR work on the influence of retinoids on membrane permeability is also described, followed by a discussion of additional ESR spin label experiments that should prove informative in the future. General Experimental Detail
Materials. Phospholipids are available from Avanti Polar Lipids (Birmingham, AL); all-trans-retinoids may be purchased from Aldrich (Milwaukee, WI). Molecular Probes, Inc. (Eugene, OR) is a source of spin labels. Preparation of Samples Multilamellar liposomes and sonicated unilamellar vesicles of phospholipids in the absence and presence of retinoids have been employed in ESR studies. Lipid mixtures are initially codissolved in chloroform. The solvent is then removed under a stream of nitrogen followed by vacuum pumping overnight. The addition of buffer and vortex mixing at temperatures at least 10° above the gel to liquid crystalline phase transition produce multilameUar liposomes. Subsequent sonication results in unilamellar vesicles. A Tekmar VC 250 Ultrasonic Cell Disruptor is suitable for this purpose. A water-cooled jacket is necessary to prevent excessive sample heating during a typical 5-min period of sonication, which is performed under nitrogen to reduce oxidation. Titanium fragments from the sonicating probe are removed by briefly spinning ( - 5 min) in a benchtop centrifuge.
Electron Spin Resonance An IBM/Bruker ER 200D X-band ESR spectrometer operating at 9.2 GHz is utilized in all our investigations. The spectrometer is interfaced and controlled by a Hewlett Packard 9816 computer system with graphics display and Winchester disk drive. The signals are detected as the first derivative of the absorption. Spectral parameters are as follows: microwave power, 12 mW; field strength, 3294 G; sweep width, 80-100 G; 14 D. Marsh, in "Membrane Spectroscopy--Molecular Biology, Biochemistry and Biophysics (E. Grell, ed.), Vol. 31, p. 51. Springer-Verlag, Berlin, 1981.
[41]
E S R OF RETINOIDS IN PHOSPHOLIPID MEMBRANES
385
sweep time, 160-200 sec; time constant, 500 msec; modulation amplitude, 1.0-2.0 G; and dataset, 1000-2000 points. Temperature is regulated within 0.1 ° by an Omega Engineering, Inc. (Stamford, CT) Model 149 Controller and monitored immediately adjacent to the sample by a copper-constantan thermocouple. Samples are contained in a capilliary tube and are approximately 25/xl in volume. Electron Spin Resonance Studies of Retinoid-Phospholipid Interactions
Basic Concepts Observation of an ESR spectrum requires the presence of an unpaired electron. In biological systems this is achieved by the introduction of a stable free radical, usually a nitroxide spin label attached to the molecule of interest. The spectrum for such a group contains three lines, the frequency (g value) and splitting (A value) of which depend on the orientation of the group with respect to the applied magnetic field.15 At sufficiently low concentrations when label-label interactions are negligible, the shape of a spectrum, in practice, is consequently determined by the anisotropy and rate of motions undergone by the spin-labeled group on the ESR time scale (10-11-10 -7 sec).
Acyl Chain Order and Fluidity Molecular ordering and dynamics within phospholipid membranes are monitored with 5-, 7-, 10-, 12-, and 16-doxylstearic acids [fl-5-, 7-, 10-, 12-, and 16-(4',4'-dimethyloxazolidinyl-N-oxyl) stearic acids] intercalated at low concentration (1 mol%) into the membrane. Although spectra simu-
0
N--O
Doxylstearic Acid lations can provide more detailed analysis, 16 information is directly derived from the spectra in a relatively simple manner. Order parameters S ~5 I. D. Campbell and R. A. Dwek, in "Biological Spectroscopy," p. 179. Benjamin/Cummings, Menlo Park, California, 1984. t6 M. Moser, D. Marsh, P. Meier, K-H. Wassmer, and G. Kothe, Biophys. J. 55, 111 (1989).
386
RECEPTORS, TRANSPORT, AND BINDING PROTEINS
[41]
and correlation times zc are calculated according to the equations S=
All-A±- C All + 2A± + 2C (1.66)
(1)
where All and A± are the apparent parallel and perpendicular hyperfine splitting parameters, the constant C = 1.4 - 0.053(Air - A±) is an empirical correction for the difference between the true and apparent values of A±, and the factor 1.66 is a solvent polarity correction factor~7; and rc = 6.5 × 10 -10
Wo[(ho/h_l)
1/2 -
1]
(2)
where W0 is the peak to peak width of the central line and ho/h-~ is the ratio of the heights of the central and high-field lines, respectively.~S Calculation of the order parameter is limited to the upper portion of the fatty acid chain (5, 7, and 10 positions), where molecular motion is sufficiently anisotropic to produce spectra for which outer and inner hyperfine extrema are discernible (Fig. 1). It is related to the amplitude of acyl chain motion at the position labeled and can take values in the range 0 -< S -< 1, the respective limits representing isotropic motion and fast axial rotation with no flexing. This may be visualized if motion of the spin label is treated as a random wobbling that is restricted in amplitude to within a cone of angle y about the normal to the membrane surface, in which case S = ½ (cos y + cos 2 T)
(3)
so that S = 0 for 3' = 90 ° and S = 1 for y = 0°) 5 The correlation time equation assumes isotropic motion and is appropriate in the lower portion of the fatty acid chain (12 and 16 positions), where molecular motion is approximately isotropic and produces spectra characteristic of high disorder (Fig. 1). As the motion is not truly isotropic, it should be realized that the ~-~values obtained are not true correlation times. They are determined by the degree of anisotropy of motion as well as by the rate, and they can only be related crudely to microviscosity or fluidity within the membrane. The effect of 0-20 tool% incorporation of all-trans-retinol, retinal, and retinoic acid on order parameters measured for 7- and10-doxylstearic acid (1 mol%) intercalated into multilamellar liposomes of 1% (w/v) DPPC (dipalmitoylphosphatidylcholine) in 20 mM phosphate/1 mM EDTA buffer (pH 7.5) at 50 ° 12is plotted in Fig. 2. The concentration dependence at the 7 position demonstrates that retinol and retinal slightly increase 17 B. J. Gaffney, in "Spin Labeling: Theory and Applications" (L. J. Berliner, ed.), p. 567. Academic Press, New York, 1976. is A. Keith, G. Bulfield, and W. Snipes, Biophys. J. 10, 618 (1970).
[41]
E S R OF RETINOIDS IN PHOSPHOLIPID MEMBRANES
387
a
o
I i
:~ 2 A.I_~: IJ o~
,! I
2All
.,,
>:
b o o ~ b . .
-i
J
!
W0 FIG. 1. Typical ESR spectra for spin-labeled stearic acids intercalated into phospholipid membranes. (a) Order parameter S calculation in the upper region of the acyl chain (5, 7, and 10 positions). (b) Correlation time ~-¢calculation in the lower region of the acyl chain (12 and 16 positions).
388
RECEPTORS, TRANSPORT,AND BINDINGPROTEINS
[41]
0.50 7-doxyl stearic acld
0.40
S 0.30
~O-doxyl ~tearic acid
0.20
0.']0 I
I
0
I
I
10
Mol%
I
20
retinoid
FIG. 2. Dependenceon all-trans-retinoid concentrationof order parameters S for 7- and 10-doxylstearicacids (1 mol%)intercalatedinto 1% w/v multilamellardispersions of DPPC in 20 mM phosphate/1 mM EDTA buffer (pH 7.5) at 50°: ©, retinol; [] retinal; and A, retinoic acid. order within the membrane, whereas retinoic acid causes disordering by as much as 14% at 20 mol% concentration. Similar behavior is seen higher up the chain at the 5 position. In contrast, at the 10 position greater order is produced by all three retinoids. The increase is approximately 22% in each case when 20 mol% is present. Qualitatively, the same trend persists further down the chain at the 12 and 16 positions, where correlation times are increased by retinol, retinal, and retinoic acid. The strongly hydrophilic nature of the carboxylic group was proposed to be responsible for the different profile of effect on acyl chain motion produced by retinoic acid. ~z It was argued that retinoic acid would be placed higher within the membrane than retinol or retinal. The consequent disturbance to molecular packing at the membrane surface would thus lead to increased disorder in the upper region of the chain. This is as opposed to the small perturbation near the aqueous interface anticipated owing to a lower location within the membrane for the less strongly hydrophilic alcohol group of retinol or aldehyde group of retinal. Experimental evidence in favor of the explanation is offered by ESR study of 5doxylstearic acid in DPPC membranes, which shows that decanol has a negligible effect on order whereas decanoic acid decreases order. 12 The
[41]
E S R OF RETINOIDS IN PHOSPHOLIPID MEMBRANES
389
bulky hydrophobic cyclohexene group of all three retinoids, however, would be expected to locate and hinder molecular motion toward the center of the membrane. ESR experiments on the influence of all-trans-retinoids on acyl chain order and fluidity within unsaturated DOPC (dioleoylphosphatidylcholine) membranes have also been performed. ~ They confirm that retinol and retinal restrict acyl chain motion in the lower portion of the chain and have little effect in the upper portion, whereas retinoic acid similarly restricts acyl chain motion in the lower portion of the chain but reduces order in the upper portion. Interestingly, the disordering produced by retinoic acid in DOPC membranes is much smaller at 35 than 45°. This is shown for the 5 position in Fig. 3. Perhaps the same trend continues at lower temperatures until retinoic acid decreases order to an even smaller or negligible extent. If so, it might be speculated that the disruption to acyl chain packing produced by the bend associated with the cis double bond (A9) of the oleic fatty acid chain creates a space into which the cyclohexene group is constrained to fit. The result this could have on the depth of penetration of retinoic acid into the membrane is illustrated by Fig. 4, where the cyclohexene group is placed in the space beneath the 0.55 35'C
0.54
0.53
S 0.52
0.51
0.50
I
0
I
I
~0
I
"1
2O
No1% r e t i n o i d
FIG. 3. Dependence on all-trans-retinoic acid concentration of order parameters S for 5doxylstearic acid (1 mol%) intercalated into 1% (w/v) multilamellar dispersions of DOPC in 20 mM phosphate/1 mM EDTA buffer (pH 7.5) at 35 and 45°.
390
RECEPTORS, TRANSPORT, AND BINDING PROTEINS
[41]
FIG. 4. Schematic representation of the position of retinoic acid (left) relative to an oleic acid acyl chain (right).
bend at the double bond in an otherwise all-trans-oleic acid chain. As can be seen, a lower location within the membrane would be imposed on the retinoic acid so that its carboxylic group would no longer protrude into the aqueous interface. Thus, the distinction in effect on molecular ordering of retinoic acid with respect to retinol and retinal would be lost. A reservation concerning the use of spin-labeled fatty acids as probes of membrane order and fluidity should be mentioned here. The bulky nitroxide moiety constitutes a perturbation. ~9 ESR data consequently 19 M. G. Taylor and I. C. P. Smith, Biochim. Biophys. Acta 733, 256 (1983).
[41]
E S R OF RETINOIDS IN PHOSPHOLIPID MEMBRANES
391
must be considered qualitative, although the direction of effect (e.g., increased order) detected is usually correct. In view of this, although quantitative differences are expected, the discrepancy between the changes arising from retinoids seen by ESR and in a recent 2H NMR (deuterium nuclear magnetic resonance) study of DPPC membranes 13 is surprising. The latter study concluded that addition of 33 mol% all-transretinoic acid causes a slight increase in order and that the same concentration of retinol decreases order throughout the membrane.
Permeability Ascorbate reduction methods enable measurement of the permeability of membranes to water-soluble spin label molecules. 2° A typical protocol for observation of uptake of the cation TEMPOcholine (2,2,6,6-tetramethylpiperidinyl-l-oxycholine) into sonicated unilamellar vesicles is described here. Initially (time t = 0), equal volumes (1.5 ml) of vesicles [5% (w/v) phospholipid] and TEMPOcholine (6 mM) in 0.1 M KCI/10 mM Tris
'--C2CH2OH C1-
TEMPOcholine Chloride buffer (pH 7.5) are mixed as a master sample, which is subsequently maintained at the desired temperature. Aliquots (50 tzl) are removed after various times intervals (t = 0, 20, 40 . . . . . x min) and immediately mixed with 10 ~1 of 0.35 M ascorbate solution. The ascorbate reduces, hence eliminating the ESR signal from, TEMPOcholine remaining outside the vesicles. Measurement of the intensity (peak to peak height) of the low-field line in the ESR spectrum for each aliquot then reveals the time evolution of the spin label leaking into the vesicles. This consists of a signal intensity that increases with time according to the rate of permeability toward an asymptotic limiting value determined by the internal volume of the vesicles. To minimize ascorbate and TEMPOcholine leakage in a given aliquot, mixing with the ascorbate and collection of the spectra are performed at 0°. Control aliquots without ascorbate are run simultaneously to facilitate signal intensity normalization. 2o D. Marsh, A. Watts, and P. F. Knowles, Biochemistry 15, 3570 (1976).
392
RECEPTORS, TRANSPORT, AND BINDING PROTEINS
[41]
0 6 0
-]-%
0 0 0 0 0
I
0
I
20
Time
I
I
40
I
I
60
I
I
BO
(m J. n.)
FIG. 5. Leakage of TEMPOcholine into vesicles (fractional intensity 1% of ESR signal from TEMPOcholine inside vesicles) versus time for DMPC (O) and DMPC/10 mol% retinoic acid (A) vesicles at 37°.
The results of a preliminary investigation of membrane permeability to TEMPOcholine are shown in Fig. 5 for DMPC (dimyristoylphosphatidylcholine) vesicles pepared with and without I0 mol% all-trans-retinoic acid. 2~ The graph follows uptake of the cation into the vesicles at 37°, as represented by the variation with time of the fractional intensity (1%) of the spin label signal remaining after ascorbate reduction. The slope of the curve is clearly greater when retinoic acid is present, which supports previous reports of retinoid associated enhancement of membrane permeability. Future Directions
There is plenty of scope to extend current ESR spin label work on molecular ordering and dynamics within the interior of model membranes containing retinoids. Natural membranes are comprised of a vast array of phospholipid and acyl chain constituents. Mixed membranes with PE (phosphatidylethanolamine) are of particular interest owing to Schiff base formation by retinal, 22 while the apparent health benefits of fish oils 23 21 C. A. Langsford, W. Stillwell, and S. R. Wassail, unpublished results (1987). 22 H. Shichi and R. L. Somers, J. Biol. Chem. 249, 6570 (1974). 23 A. P. Simopoulos, R. R. Kifer, and R. E. Martin (eds.), "Health Effects of Polyunsaturated Fatty Acids in Seafoods." Academic Press, New York, 1986.
[41]
E S R OF RETINOIDS IN PHOSPHOLIPID MEMBRANES
393
suggest that polyunsaturated membranes are worthy of attention. Our preliminary measurement of inc.,,ased permeability to TEMPOcholine in the presence of retinoic acid furthermore encourages studies with all three retinoids. Other aspects of the interaction of retinoids with phospholipid membranes are accessible to future investigation by ESR. The application of ESR spin label methods to study membrane fusion, a process that has been linked to vitamin A , 24'25 has been reviewed. 26 Importantly, mxing of both aqueous and lipid environments may be monitored. The water-soluble spin-label TEMPO (2,2,6,6-tetramethylpiperidinyl-l-oxy) manifests a marked increase in membrane solubility at the gel to liquid crystalline phase transition 27 and may be exploited to determine the influence of retinoids on phase behavior in aqueous phospholipid dispersions. Specifically, the dependence on temperture is measured for the intensities of the two components, corresponding to TEMPO dissolved in water and membrane, in the high-field line of the ESR spectrum. Rates of lateral diffusion and flip-flop within phospholipid membranes may also be sensitive to the presence of retinoids and are cited as final examples of potential ESR work. Estimation of the former quantity relies on the dominant contribution exchange broadening makes to ESR spectra for membranes containing over 1 tool% spin-labeled fatty acid or phospholipid. 2s Analysis of the spectral broadening as a function of spin label concentration yields the diffusion coefficient. 29 Measurement of the flip-flop of head group spinlabeled PC (l,2-dipalmitoyl-sn-glycero-3-phosphorylTEMPOcholine) across the membrane of unilamellar vesicles entails an ascorbate reduction procedure. 3° The transfer of spin-labeled molecules from the inner to outer vesicle layer is observed following initial abolition of the ESR signal from the outside layer. Conclusion The intention of this chapter is to summarize previous ESR spin label work on phospholipid-retinoid interactions and to indicate possible aver24 p. Dunham, P. Babiarz, A. Israel, A. Zerial, and G. Weissmann, Proc. Natl. Acad. Sci. U.S.A. 74, 1580 0977)° 25 A. H. Goodhall, D. Fisher, and J. A. Lucy, Biochim. Biophys. Acta 595, 1 (1980). 26 H. M. McConneU, in "Spin Labeling: Theory and Applications" (L. J. Berliner, ed), p. 525. Academic Press, New York, 1976. 27 E. J. Shimshick and H. M. McConnell, Biochemistry 12, 235t (1973). 2s p. Devaux, C. J. Scandella, and H. M. McConnell, J. Magn. Reson. 9, 474 (1973). 29 E. Sackmann, H. Traiible, H.-J. Galla, and P. Overath, Biochemistry 12, 5360 (1973). 30 R. Kornberg and H. M. McConnell, Biochemistry 10, l l l l (1971).
394
RECEPTORS, TRANSPORT, AND B I N D I N G PROTEINS
[42]
nues for further research. The aim of these studies is to contribute toward an eventual definition of a relationship between the molecular structure of vitamin A and its physiological role. The current description that arises from ESR studies establishes a distinction between the response of phospholipid membranes to all-trans-retinoic acid and to all-trans-retinol and retinal. All three retinoids restrict acyl chain motion to a similar extent approaching the center of the membrane, but toward the surface of the membrane retinol and retinal are a negligible perturbation to order whereas retinoic acid causes disordering. 1°-12 Consistent with this difference in behavior, greater enhancement of membrane permeability is measured with retinoic acid than retinol or retinal. 12In the context of retinoid toxicity, it is thus noted that retinoic acid induces hypervitaminosis A more effectively than retinol. 31 Acknowledgments It is a pleasure to thank Marvin D. Kemplefor use of the ESR spectrometer, Fritz W. Kleinhans and Timothy M. Phelps for development of spectral collection and analysis routines, Carol A. Langsfordfor performingexperiments, MargoPage for typingthe manuscript, and Ellen Chernofffor photographicassistance. 31 G. A. J. Pitt, Proc. Nutr. Soc. 42, 43 (1983).
[42] T r a n s f e r o f R e t i n o l f r o m R e t i n o l - B i n d i n g P r o t e i n C o m p l e x to L i p o s o m e s a n d across L i p o s o m a l M e m b r a n e s
By G O ~ N FEX and G v n v o g JOHANNESSON Introduction The mechanism(s) of cellular uptake of retinol from its complex with plasma retinol-binding protein (RBP) and the subsequent intracellular distribution of retinol are largely unknown. According to the current view, retinol is taken up into cells after the retinol-RBP complex has interacted with a cell surface receptor for retinol-binding protein. The nature of this receptor is unknown. Using indirect methods the receptor has been demonstrated on bovine pigment epithelial cells, ~ monkey intestinal cells, 2 I j. Heller, J. Biol. Chem. 250, 3613 (1975). 2 L. R a s k a n d P. A. P e t e r s o n , J. Biol. Chem. 251, 6360 (1976).
METHODS IN ENZYMOLOGY, VOL. 189
Copyright © 1990by Academic Press, Inc. All fights of reproduction in any form reserved.
E S R OF RETINOIDS IN PI-IOSPHOLIPID MEMBRANES
383
[41] I n t e r a c t i o n s o f R e t i n o i d s w i t h P h o s p h o l i p i d M e m b r a n e s : Electron Spin Resonance
By STEPHEN R. WASSALL and WILLIAM STILLWELL Introduction Retinoids are essential for the maintenance of health. ~Their amphiphilic nature suggests that they will locate within the phospholipid bilayer of membranes, which may constitute a site of action. In addition, the toxicity of high doses of retinoids poses a serious problem to their claimed efficacy in treating a number of cancers. 2 Membrane disruption may be a cause. Several studies have demonstrated that retinoids affect the properties of membranes. Enhanced permeability to nonelectrolytes and ions on incorporation of retinoids into phospholipid membranes has been extensively reported. 3-5 Calorimetric and optical methods have shown that alltrans-retinol, retinal, and retinoic acid broaden the gel to liquid crystalline phase transition and depress its onset temperature in PC (phosphatidylcholine) bilayers. 4,6 Reduced microviscosities in rat erythrocytes and mouse fibroblasts caused by retinoids have been measured by fluorescence polarization, 7,8 whereas magnetic resonance techniques have detected retinoid-associated changes in molecular ordering and dynamics within phospholipid model membranes. 9-13 Further work, however, is required to establish a clear picture. In this chapter, the application of ESR (electron spin resonance) spin label techniques as a method of assay for the interactions of retinoids with 1 L. M. DeLuca and S. S. Shapiro (eds.), Ann. N.Y. Acad. Sci. 359 (1981). 2 D. F. Birt, Proc. Soc. Exp. Biol. Med. 183, 311 (1986). 3 W. Stillwell and M. Ricketts, Biochem. Biophys. Res. Comrnun. 97, 148 (1980). 4 W. Stillwell, M. Ricketts, H. Hudson, and S. Nahmias, Biochim. Biophys. Acta 688, 653 (1982). 5 W. Stillwell and L. Bryant, Biochim. Biophys. Acta 731, 483 (1983). 6 S. R. Wassail, W. Stillwell, and M. Zeldin, Proc. Indiana Acad. Sci. 95, 149 (1986). 7 R. G. Meeks, D. Zaharevitz, and R. F. Chen, Arch. Biochem. Biophys. 207, 141 (1981). 8 A. M. Jetten, R. G. Meeks, and L. M. DeLuca, Ann. N.Y. Acad. Sci. 359, 398 (1981). 9 S. P. Verma, H. Schneider, and I. C. P. Smith, Arch. Biochem. Biophys. 162, 48 (1974). l0 S. R. Wassail and W. Stiilwell, Bull. Magn. Reson. 9, 85 (1987). N C. A. Langsford, M. R. Albrecht, T. M. Phelps, W. Stillwell, and S. R. Wassail, Biophys. J. 51, 239a (1987). 12 S. R. Wassall, T. M. Phelps, M. R. Albrecht, C. A. Langsford, and W. Stillwell, Biochim. Biophys. Acta 939, 393 (1988). i3 H. DeBoeck and R. Zidovetzki, Biochim. Biophys. Acta 946, 244 (1988).
METHODS IN ENZYMOLOGY, VOL. 189
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
384
RECEPTORS, TRANSPORT, AND BINDING PROTEINS
[41]
phospholipid membranes is reviewed. The utility of the approach in membrane research is well documented. 14This chapter focuses on recent studies of the effects of retinoids on order and fluidity within phospholipid model membranes, which demonstrated a distinction between the effects of all-trans-retinoic acid and those of all-trans-retinol and retinal. 10-12Preliminary ESR work on the influence of retinoids on membrane permeability is also described, followed by a discussion of additional ESR spin label experiments that should prove informative in the future. General Experimental Detail
Materials. Phospholipids are available from Avanti Polar Lipids (Birmingham, AL); all-trans-retinoids may be purchased from Aldrich (Milwaukee, WI). Molecular Probes, Inc. (Eugene, OR) is a source of spin labels. Preparation of Samples Multilamellar liposomes and sonicated unilamellar vesicles of phospholipids in the absence and presence of retinoids have been employed in ESR studies. Lipid mixtures are initially codissolved in chloroform. The solvent is then removed under a stream of nitrogen followed by vacuum pumping overnight. The addition of buffer and vortex mixing at temperatures at least 10° above the gel to liquid crystalline phase transition produce multilameUar liposomes. Subsequent sonication results in unilamellar vesicles. A Tekmar VC 250 Ultrasonic Cell Disruptor is suitable for this purpose. A water-cooled jacket is necessary to prevent excessive sample heating during a typical 5-min period of sonication, which is performed under nitrogen to reduce oxidation. Titanium fragments from the sonicating probe are removed by briefly spinning ( - 5 min) in a benchtop centrifuge.
Electron Spin Resonance An IBM/Bruker ER 200D X-band ESR spectrometer operating at 9.2 GHz is utilized in all our investigations. The spectrometer is interfaced and controlled by a Hewlett Packard 9816 computer system with graphics display and Winchester disk drive. The signals are detected as the first derivative of the absorption. Spectral parameters are as follows: microwave power, 12 mW; field strength, 3294 G; sweep width, 80-100 G; 14 D. Marsh, in "Membrane Spectroscopy--Molecular Biology, Biochemistry and Biophysics (E. Grell, ed.), Vol. 31, p. 51. Springer-Verlag, Berlin, 1981.
[41]
E S R OF RETINOIDS IN PHOSPHOLIPID MEMBRANES
385
sweep time, 160-200 sec; time constant, 500 msec; modulation amplitude, 1.0-2.0 G; and dataset, 1000-2000 points. Temperature is regulated within 0.1 ° by an Omega Engineering, Inc. (Stamford, CT) Model 149 Controller and monitored immediately adjacent to the sample by a copper-constantan thermocouple. Samples are contained in a capilliary tube and are approximately 25/xl in volume. Electron Spin Resonance Studies of Retinoid-Phospholipid Interactions
Basic Concepts Observation of an ESR spectrum requires the presence of an unpaired electron. In biological systems this is achieved by the introduction of a stable free radical, usually a nitroxide spin label attached to the molecule of interest. The spectrum for such a group contains three lines, the frequency (g value) and splitting (A value) of which depend on the orientation of the group with respect to the applied magnetic field.15 At sufficiently low concentrations when label-label interactions are negligible, the shape of a spectrum, in practice, is consequently determined by the anisotropy and rate of motions undergone by the spin-labeled group on the ESR time scale (10-11-10 -7 sec).
Acyl Chain Order and Fluidity Molecular ordering and dynamics within phospholipid membranes are monitored with 5-, 7-, 10-, 12-, and 16-doxylstearic acids [fl-5-, 7-, 10-, 12-, and 16-(4',4'-dimethyloxazolidinyl-N-oxyl) stearic acids] intercalated at low concentration (1 mol%) into the membrane. Although spectra simu-
0
N--O
Doxylstearic Acid lations can provide more detailed analysis, 16 information is directly derived from the spectra in a relatively simple manner. Order parameters S ~5 I. D. Campbell and R. A. Dwek, in "Biological Spectroscopy," p. 179. Benjamin/Cummings, Menlo Park, California, 1984. t6 M. Moser, D. Marsh, P. Meier, K-H. Wassmer, and G. Kothe, Biophys. J. 55, 111 (1989).
386
RECEPTORS, TRANSPORT, AND BINDING PROTEINS
[41]
and correlation times zc are calculated according to the equations S=
All-A±- C All + 2A± + 2C (1.66)
(1)
where All and A± are the apparent parallel and perpendicular hyperfine splitting parameters, the constant C = 1.4 - 0.053(Air - A±) is an empirical correction for the difference between the true and apparent values of A±, and the factor 1.66 is a solvent polarity correction factor~7; and rc = 6.5 × 10 -10
Wo[(ho/h_l)
1/2 -
1]
(2)
where W0 is the peak to peak width of the central line and ho/h-~ is the ratio of the heights of the central and high-field lines, respectively.~S Calculation of the order parameter is limited to the upper portion of the fatty acid chain (5, 7, and 10 positions), where molecular motion is sufficiently anisotropic to produce spectra for which outer and inner hyperfine extrema are discernible (Fig. 1). It is related to the amplitude of acyl chain motion at the position labeled and can take values in the range 0 -< S -< 1, the respective limits representing isotropic motion and fast axial rotation with no flexing. This may be visualized if motion of the spin label is treated as a random wobbling that is restricted in amplitude to within a cone of angle y about the normal to the membrane surface, in which case S = ½ (cos y + cos 2 T)
(3)
so that S = 0 for 3' = 90 ° and S = 1 for y = 0°) 5 The correlation time equation assumes isotropic motion and is appropriate in the lower portion of the fatty acid chain (12 and 16 positions), where molecular motion is approximately isotropic and produces spectra characteristic of high disorder (Fig. 1). As the motion is not truly isotropic, it should be realized that the ~-~values obtained are not true correlation times. They are determined by the degree of anisotropy of motion as well as by the rate, and they can only be related crudely to microviscosity or fluidity within the membrane. The effect of 0-20 tool% incorporation of all-trans-retinol, retinal, and retinoic acid on order parameters measured for 7- and10-doxylstearic acid (1 mol%) intercalated into multilamellar liposomes of 1% (w/v) DPPC (dipalmitoylphosphatidylcholine) in 20 mM phosphate/1 mM EDTA buffer (pH 7.5) at 50 ° 12is plotted in Fig. 2. The concentration dependence at the 7 position demonstrates that retinol and retinal slightly increase 17 B. J. Gaffney, in "Spin Labeling: Theory and Applications" (L. J. Berliner, ed.), p. 567. Academic Press, New York, 1976. is A. Keith, G. Bulfield, and W. Snipes, Biophys. J. 10, 618 (1970).
[41]
E S R OF RETINOIDS IN PHOSPHOLIPID MEMBRANES
387
a
o
I i
:~ 2 A.I_~: IJ o~
,! I
2All
.,,
>:
b o o ~ b . .
-i
J
!
W0 FIG. 1. Typical ESR spectra for spin-labeled stearic acids intercalated into phospholipid membranes. (a) Order parameter S calculation in the upper region of the acyl chain (5, 7, and 10 positions). (b) Correlation time ~-¢calculation in the lower region of the acyl chain (12 and 16 positions).
388
RECEPTORS, TRANSPORT,AND BINDINGPROTEINS
[41]
0.50 7-doxyl stearic acld
0.40
S 0.30
~O-doxyl ~tearic acid
0.20
0.']0 I
I
0
I
I
10
Mol%
I
20
retinoid
FIG. 2. Dependenceon all-trans-retinoid concentrationof order parameters S for 7- and 10-doxylstearicacids (1 mol%)intercalatedinto 1% w/v multilamellardispersions of DPPC in 20 mM phosphate/1 mM EDTA buffer (pH 7.5) at 50°: ©, retinol; [] retinal; and A, retinoic acid. order within the membrane, whereas retinoic acid causes disordering by as much as 14% at 20 mol% concentration. Similar behavior is seen higher up the chain at the 5 position. In contrast, at the 10 position greater order is produced by all three retinoids. The increase is approximately 22% in each case when 20 mol% is present. Qualitatively, the same trend persists further down the chain at the 12 and 16 positions, where correlation times are increased by retinol, retinal, and retinoic acid. The strongly hydrophilic nature of the carboxylic group was proposed to be responsible for the different profile of effect on acyl chain motion produced by retinoic acid. ~z It was argued that retinoic acid would be placed higher within the membrane than retinol or retinal. The consequent disturbance to molecular packing at the membrane surface would thus lead to increased disorder in the upper region of the chain. This is as opposed to the small perturbation near the aqueous interface anticipated owing to a lower location within the membrane for the less strongly hydrophilic alcohol group of retinol or aldehyde group of retinal. Experimental evidence in favor of the explanation is offered by ESR study of 5doxylstearic acid in DPPC membranes, which shows that decanol has a negligible effect on order whereas decanoic acid decreases order. 12 The
[41]
E S R OF RETINOIDS IN PHOSPHOLIPID MEMBRANES
389
bulky hydrophobic cyclohexene group of all three retinoids, however, would be expected to locate and hinder molecular motion toward the center of the membrane. ESR experiments on the influence of all-trans-retinoids on acyl chain order and fluidity within unsaturated DOPC (dioleoylphosphatidylcholine) membranes have also been performed. ~ They confirm that retinol and retinal restrict acyl chain motion in the lower portion of the chain and have little effect in the upper portion, whereas retinoic acid similarly restricts acyl chain motion in the lower portion of the chain but reduces order in the upper portion. Interestingly, the disordering produced by retinoic acid in DOPC membranes is much smaller at 35 than 45°. This is shown for the 5 position in Fig. 3. Perhaps the same trend continues at lower temperatures until retinoic acid decreases order to an even smaller or negligible extent. If so, it might be speculated that the disruption to acyl chain packing produced by the bend associated with the cis double bond (A9) of the oleic fatty acid chain creates a space into which the cyclohexene group is constrained to fit. The result this could have on the depth of penetration of retinoic acid into the membrane is illustrated by Fig. 4, where the cyclohexene group is placed in the space beneath the 0.55 35'C
0.54
0.53
S 0.52
0.51
0.50
I
0
I
I
~0
I
"1
2O
No1% r e t i n o i d
FIG. 3. Dependence on all-trans-retinoic acid concentration of order parameters S for 5doxylstearic acid (1 mol%) intercalated into 1% (w/v) multilamellar dispersions of DOPC in 20 mM phosphate/1 mM EDTA buffer (pH 7.5) at 35 and 45°.
390
RECEPTORS, TRANSPORT, AND BINDING PROTEINS
[41]
FIG. 4. Schematic representation of the position of retinoic acid (left) relative to an oleic acid acyl chain (right).
bend at the double bond in an otherwise all-trans-oleic acid chain. As can be seen, a lower location within the membrane would be imposed on the retinoic acid so that its carboxylic group would no longer protrude into the aqueous interface. Thus, the distinction in effect on molecular ordering of retinoic acid with respect to retinol and retinal would be lost. A reservation concerning the use of spin-labeled fatty acids as probes of membrane order and fluidity should be mentioned here. The bulky nitroxide moiety constitutes a perturbation. ~9 ESR data consequently 19 M. G. Taylor and I. C. P. Smith, Biochim. Biophys. Acta 733, 256 (1983).
[41]
E S R OF RETINOIDS IN PHOSPHOLIPID MEMBRANES
391
must be considered qualitative, although the direction of effect (e.g., increased order) detected is usually correct. In view of this, although quantitative differences are expected, the discrepancy between the changes arising from retinoids seen by ESR and in a recent 2H NMR (deuterium nuclear magnetic resonance) study of DPPC membranes 13 is surprising. The latter study concluded that addition of 33 mol% all-transretinoic acid causes a slight increase in order and that the same concentration of retinol decreases order throughout the membrane.
Permeability Ascorbate reduction methods enable measurement of the permeability of membranes to water-soluble spin label molecules. 2° A typical protocol for observation of uptake of the cation TEMPOcholine (2,2,6,6-tetramethylpiperidinyl-l-oxycholine) into sonicated unilamellar vesicles is described here. Initially (time t = 0), equal volumes (1.5 ml) of vesicles [5% (w/v) phospholipid] and TEMPOcholine (6 mM) in 0.1 M KCI/10 mM Tris
'--C2CH2OH C1-
TEMPOcholine Chloride buffer (pH 7.5) are mixed as a master sample, which is subsequently maintained at the desired temperature. Aliquots (50 tzl) are removed after various times intervals (t = 0, 20, 40 . . . . . x min) and immediately mixed with 10 ~1 of 0.35 M ascorbate solution. The ascorbate reduces, hence eliminating the ESR signal from, TEMPOcholine remaining outside the vesicles. Measurement of the intensity (peak to peak height) of the low-field line in the ESR spectrum for each aliquot then reveals the time evolution of the spin label leaking into the vesicles. This consists of a signal intensity that increases with time according to the rate of permeability toward an asymptotic limiting value determined by the internal volume of the vesicles. To minimize ascorbate and TEMPOcholine leakage in a given aliquot, mixing with the ascorbate and collection of the spectra are performed at 0°. Control aliquots without ascorbate are run simultaneously to facilitate signal intensity normalization. 2o D. Marsh, A. Watts, and P. F. Knowles, Biochemistry 15, 3570 (1976).
392
RECEPTORS, TRANSPORT, AND BINDING PROTEINS
[41]
0 6 0
-]-%
0 0 0 0 0
I
0
I
20
Time
I
I
40
I
I
60
I
I
BO
(m J. n.)
FIG. 5. Leakage of TEMPOcholine into vesicles (fractional intensity 1% of ESR signal from TEMPOcholine inside vesicles) versus time for DMPC (O) and DMPC/10 mol% retinoic acid (A) vesicles at 37°.
The results of a preliminary investigation of membrane permeability to TEMPOcholine are shown in Fig. 5 for DMPC (dimyristoylphosphatidylcholine) vesicles pepared with and without I0 mol% all-trans-retinoic acid. 2~ The graph follows uptake of the cation into the vesicles at 37°, as represented by the variation with time of the fractional intensity (1%) of the spin label signal remaining after ascorbate reduction. The slope of the curve is clearly greater when retinoic acid is present, which supports previous reports of retinoid associated enhancement of membrane permeability. Future Directions
There is plenty of scope to extend current ESR spin label work on molecular ordering and dynamics within the interior of model membranes containing retinoids. Natural membranes are comprised of a vast array of phospholipid and acyl chain constituents. Mixed membranes with PE (phosphatidylethanolamine) are of particular interest owing to Schiff base formation by retinal, 22 while the apparent health benefits of fish oils 23 21 C. A. Langsford, W. Stillwell, and S. R. Wassail, unpublished results (1987). 22 H. Shichi and R. L. Somers, J. Biol. Chem. 249, 6570 (1974). 23 A. P. Simopoulos, R. R. Kifer, and R. E. Martin (eds.), "Health Effects of Polyunsaturated Fatty Acids in Seafoods." Academic Press, New York, 1986.
[41]
E S R OF RETINOIDS IN PHOSPHOLIPID MEMBRANES
393
suggest that polyunsaturated membranes are worthy of attention. Our preliminary measurement of inc.,,ased permeability to TEMPOcholine in the presence of retinoic acid furthermore encourages studies with all three retinoids. Other aspects of the interaction of retinoids with phospholipid membranes are accessible to future investigation by ESR. The application of ESR spin label methods to study membrane fusion, a process that has been linked to vitamin A , 24'25 has been reviewed. 26 Importantly, mxing of both aqueous and lipid environments may be monitored. The water-soluble spin-label TEMPO (2,2,6,6-tetramethylpiperidinyl-l-oxy) manifests a marked increase in membrane solubility at the gel to liquid crystalline phase transition 27 and may be exploited to determine the influence of retinoids on phase behavior in aqueous phospholipid dispersions. Specifically, the dependence on temperture is measured for the intensities of the two components, corresponding to TEMPO dissolved in water and membrane, in the high-field line of the ESR spectrum. Rates of lateral diffusion and flip-flop within phospholipid membranes may also be sensitive to the presence of retinoids and are cited as final examples of potential ESR work. Estimation of the former quantity relies on the dominant contribution exchange broadening makes to ESR spectra for membranes containing over 1 tool% spin-labeled fatty acid or phospholipid. 2s Analysis of the spectral broadening as a function of spin label concentration yields the diffusion coefficient. 29 Measurement of the flip-flop of head group spinlabeled PC (l,2-dipalmitoyl-sn-glycero-3-phosphorylTEMPOcholine) across the membrane of unilamellar vesicles entails an ascorbate reduction procedure. 3° The transfer of spin-labeled molecules from the inner to outer vesicle layer is observed following initial abolition of the ESR signal from the outside layer. Conclusion The intention of this chapter is to summarize previous ESR spin label work on phospholipid-retinoid interactions and to indicate possible aver24 p. Dunham, P. Babiarz, A. Israel, A. Zerial, and G. Weissmann, Proc. Natl. Acad. Sci. U.S.A. 74, 1580 0977)° 25 A. H. Goodhall, D. Fisher, and J. A. Lucy, Biochim. Biophys. Acta 595, 1 (1980). 26 H. M. McConneU, in "Spin Labeling: Theory and Applications" (L. J. Berliner, ed), p. 525. Academic Press, New York, 1976. 27 E. J. Shimshick and H. M. McConnell, Biochemistry 12, 235t (1973). 2s p. Devaux, C. J. Scandella, and H. M. McConnell, J. Magn. Reson. 9, 474 (1973). 29 E. Sackmann, H. Traiible, H.-J. Galla, and P. Overath, Biochemistry 12, 5360 (1973). 30 R. Kornberg and H. M. McConnell, Biochemistry 10, l l l l (1971).
394
RECEPTORS, TRANSPORT, AND B I N D I N G PROTEINS
[42]
nues for further research. The aim of these studies is to contribute toward an eventual definition of a relationship between the molecular structure of vitamin A and its physiological role. The current description that arises from ESR studies establishes a distinction between the response of phospholipid membranes to all-trans-retinoic acid and to all-trans-retinol and retinal. All three retinoids restrict acyl chain motion to a similar extent approaching the center of the membrane, but toward the surface of the membrane retinol and retinal are a negligible perturbation to order whereas retinoic acid causes disordering. 1°-12 Consistent with this difference in behavior, greater enhancement of membrane permeability is measured with retinoic acid than retinol or retinal. 12In the context of retinoid toxicity, it is thus noted that retinoic acid induces hypervitaminosis A more effectively than retinol. 31 Acknowledgments It is a pleasure to thank Marvin D. Kemplefor use of the ESR spectrometer, Fritz W. Kleinhans and Timothy M. Phelps for development of spectral collection and analysis routines, Carol A. Langsfordfor performingexperiments, MargoPage for typingthe manuscript, and Ellen Chernofffor photographicassistance. 31 G. A. J. Pitt, Proc. Nutr. Soc. 42, 43 (1983).
[42] T r a n s f e r o f R e t i n o l f r o m R e t i n o l - B i n d i n g P r o t e i n C o m p l e x to L i p o s o m e s a n d across L i p o s o m a l M e m b r a n e s
By G O ~ N FEX and G v n v o g JOHANNESSON Introduction The mechanism(s) of cellular uptake of retinol from its complex with plasma retinol-binding protein (RBP) and the subsequent intracellular distribution of retinol are largely unknown. According to the current view, retinol is taken up into cells after the retinol-RBP complex has interacted with a cell surface receptor for retinol-binding protein. The nature of this receptor is unknown. Using indirect methods the receptor has been demonstrated on bovine pigment epithelial cells, ~ monkey intestinal cells, 2 I j. Heller, J. Biol. Chem. 250, 3613 (1975). 2 L. R a s k a n d P. A. P e t e r s o n , J. Biol. Chem. 251, 6360 (1976).
METHODS IN ENZYMOLOGY, VOL. 189
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