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The influence of cholesterol on synaptic fluidity and dopamine uptake
Brain Research Bulletin, Vol. 22, pp. 431437.
0 Pergamon Press plc, 1989. Printed in the U.S.A.
0361-9230/89 $3.00 + .OO
The Influence of Cholesterol on Synaptic Fluidity and Dopamine Uptake’ PATRICIA
A. MAGUIRE2
AND MARY
J. DRUSE3
Department of Biochemistry and Biophysics Loyola University Stritch School of Medicine, Maywood, Received MAGUIRE,
P. A. AND M. J. DRUSE.
The influence
1 July
of cholesterol
IL 601.53
1988 on
synapticfluidity
and dopamine
uptake.
BRAIN
RES BULL 22(2) 431-437, 1989.-The present study examined how the synaptic cholesteroVphospholipid ratio influences membrane fluidity and a transmembrane process of the dopaminergic system, dopamine uptake. Fluorescence polarization of DPH was used as a measure of membrane fluidity. The cholesterol content of synaptosomal and synaptic plasma membranes was altered using a lipid transfer protein. The results of the present study demonstrate that the transmembrane process of dopamine uptake may be inhibited by an elevated synaptic cholesterol/phospholipid ratio, which correlates with increased fluorescence polarization. Specifically, a l&30% increase in the cholesterol/phospholipid ratio resulted in an -20% increase in fluorescence polarization, a 3- to 6-fold increase in K,, a 4-fold increase in V,,, , and a marked decrease in the V,,,,,/K,,, (the first order rate constant for dopamine uptake at low substrate concentrations). Fluidity
Dopamine
Uptake
Synaptic plasma membranes
THERE is evidence that as an animal matures and ages, the cholesteroYphospholipid ratio of synaptic membranes increases and membrane fluidity decreases (1, 13, 15, 26, 31). These synaptic membrane changes are accompanied, temporally, by alterations in the components of several neurotransmitter systems [reviewed in (30)]. Since many membrane-associated functions are markedly influenced by membrane lipids and fluidity [examples given in (2, 27, 28, 31, 37)], it is possible that at least some of the age-associated changes of the neurotransmitter systems are related to alterations in the cholesterol/phospholipid ratio and fluidity of synaptic membranes. In an attempt to investigate this hypothesis, we examined the influence of the synaptic cholesterol/phospholipid ratio on the fluorescence polarization of l,ddiphenyl-1,3,5-hexatriene (DPH) and on a membrane-associated function of the dopaminergic system. The dopaminergic system was studied because several components of this system appear to be altered by aging (8, 18, 22, 24, 25, 38). Specifically, we examined the influence of the cholesterol/ phospholipid ratio on the uptake of dopamine. Dopamine uptake was studied since such a transmembrane process might be expected to be sensitive to changes in membrane composition and fluidity. The cholesterol content was altered using a lipid transfer protein (28) which facilitates the rapid transport of cholesterol between synaptic membranes and liposomes.
Synaptosomes
Cholesterol
METHOD Preparation
of Liposomes
Three types of liposomes were used in transfer experiments. One type of liposome was used to determine the activity of the lipid transfer protein by assessing the ability of the protein to transfer cholesterol from liposomes to mitochondria. These liposomes were prepared using 1 mg phosphatidylcholine (PC) per ml and 1 mole cholesterol per mole of PC and contained 0.25 @Zi each of [WI-cholesterol (Amersham, Arlington Heights, IL) and PHI-triolein (ICN Radiochemicals, Irvine, CA) as transferrable and nontransferrable markers, respectively (28). The other two types of liposomes were used in experiments in which synaptosomes and synaptic plasma membranes @PM) were either loaded with or depleted of cholesterol. That is, they were used in experiments in which the lipid transfer protein facilitated a net transfer of cholesterol from liposomes to synaptosomes or SPM (cholesterol loading) or from synaptosomes or SPM to liposomes (cholesterol depletion). For cholesterol depletion studies, liposomes were prepared using 10 mg egg PC per ml. Those to be used for cholesterol loading were prepared with 5 mg PC/ml and a cholesterol/ phospholipid molar ratio of - 1.7. Both of the latter types of liposomes also contained 0.25 &i of the nontransferrable marker [“‘Cl-tripalmitin (Amersham, Arlington Height, IL). To prepare liposomes, lipids were mixed in chloroform and
‘This research was submitted in partial fulfillment of the Ph.D. requirements of Loyola University of Chicago (P.A.M.). *Present address: Department of Biology, University of California, Riverside, CA 92521. 3Requests for reprints should be addressed to Mary J. Druse Manteuffel.
431
MAGUIRE dried, under a stream of nitrogen, to a thin film on the sides of an acid-washed tube. Lipid films were dispersed in 0.32 M sucrose, 5 mM Tris, pH 7.0, by extensive mixing on a Vortex mixer. The mixed samples were then sonicated until the lipid mixture was translucent. Sonicated liposomes were centrifuged at 27,000 g for 60 minutes and used within 3 hours of preparation. isolation of Lipid Transfer Protein
The nonspecific lipid transfer protein was partially purified from beef liver, obtained from Aurora Packing Company (North Aurora, IL), by a modification of previous procedures of (5,28). All procedures were done at 4°C. A 3% (w/v) homogenate was prepared in a buffer containing 0.25 M sucrose, 1 mM EDTA, 50 mM Tris-HCl, and 0.0% (w/v) sodium azide, pH 7.4. The homogenate was centrifuged at 13,000 g for 30 minutes. The pH of the supematant was adjusted to 5.1 with HCl and mixed for 1 hour prior to centrifugation at 13,000 g for 20 minutes. The pH of the clear supematant (5.1 supematant) was returned to 7.4 with NaOH. Aliquots were stored at -20°C for up to 6 months. Protein in the 5.1 supematant was precipitated with ammonium sulfate between 4% and 90% saturation. The resulting pellet was suspended in a buffer containing 25 mM sodium phosphate, 10 mM 2-mercaptoethanol and 0.0% (w/v) sodium azide, pH 8.1, and dialyzed extensively against a solution containing 5 mM sodium phosphate, 5 mM 2mercaptoethanol, 0.0% (w/v) sodium azide, pH 8.1. The dialysate was applied to a Sephadex CM-C25 (Pharmacia, Inc., Uppsala, Sweden) ion exchange column and washed extensively with dialysis buffer. Transfer activity was eluted with a solution containing 25 mM sodium phosphate, 45 mM NaCl, 5 mM 2-mercaptoethanol and 0.0% (w/v) sodium azide, pH 8.1 (elution buffer). Transfer activity was stable in elution buffer for 1 month at 4°C. Prior to use, the transfer protein was dialyzed against the elution buffer without azide and concentrated 5- to IO-fold by ultrafiltration through an Amicon cell, equipped with a Diaflo Y-M5 membrane (Amicon Corp., Danvers, MA). The ability of the purified proteins to transfer cholesterol between liposomes and membranes was assessed by the standard procedure (28) which uses beef heart mitochondria (4) as the cholesterol acceptor. Mitochondria are typically used as an acceptor because of their low cholesterol content and their ease of preparation. Isolation of Synaptosomes Membranes (SPM)
and Synaptic Plasma
Synaptosomes and SPM were isolated from the brains of 40-day-old male Sprague-Dawley rats (Holtzman, Madison, WI) (4) and used immediately thereafter in cholesterol loading and depletion experiments. Normal and lipid-modified synaptosomes were used to assess dopamine uptake. However, SPM were used in fluorescence polarization studies rather than synaptosomes in order to minimize the partitioning of the hydrophobic fluorescent probes into membranes other than the synaptic plasma membrane (e.g., synaptosomal mitochondria and vesicles). The importance of using purified plasma membranes as a means of minimizing membrane heterogeneity has been stressed previously (29,34). Although synaptosomal and SPM preparations are not identical, one should be able to cautiously extrapolate the results from one to the other. Aliquots of control and modified synaptosomes and SPM
AND DRUSE
were stored at -80°C prior to analysis of protein (21), phospholipid (12), and cholesterol (Sigma Kit 351, Sigma, St. Louis, MO). Fifty micrograms of SPM protein were used routinely in the fluorescence polarization studies. Fifty micrograms of SPM protein corresponds to -37 pg phospholipid and -10 ,ug cholesterol in control SPM. In a comparable sample of modified SPM the phospholipid content was not significantly altered, however, the cholesterol content ranged from -6.5 to -16.7 pg. Two to three hundred micrograms of synaptosomal protein were used in dopamine uptake experiments. Lipid Transfer Between Liposomes
and Synaptosomes
Synaptosomes or SPM (1 mg protein in 50 ~1 buffered 0.32 M sucrose) were preincubated for 10 minutes at 32°C with 200 ~1 of concentrated transfer protein in elution buffer, 80 ~1 of 1.8 M sucrose, and 170 ~1 of deionized water (final sucrose concentration = 0.32 M). The 200 ~1 of concentrated transfer protein contained from 0.1 to 0.4 mg of protein. The reaction was initiated by the addition of 500 ~1 of liposomes, and terminated after either 30 (synaptosomes) or 60 minutes @PM) by centrifugation at 106,000 g for 40 minutes through a layer 0.5 M sucrose (28). Pellets were rinsed and suspended in the appropriate buffer for subsequent analyses. Control and modified synaptosomes were gently suspended in modified Krebs-Henseleit buffer (118 mM NaCl, 26 mM NqHPQ, 6 mM NaH,P04, 4.7 mM KCl, 1.8 CaCl,, 1.2 mM MgS04, 1.3 mM EDTA, 5.6 mM D-glucose, 2 FM ascorbic acid, 10 PM pargyline, pH 7.4) while SPM were suspended in a solution containing 0.32 M sucrose and 5 mM Tris, pH 7.0. Dopamine
Uptake
Synaptosomal uptake of dopamine was assayed by a modification of a standard procedure (9). Triplicate tubes were prepared which each contained 10-15 nM rH]dopamine (New England Nuclear, Boston, MA) and O-5000 nM unlabeled dopamine in 900 ~1 of the Krebs buffer. The reaction was initiated by the addition of 100 ~1 aliquots containing 200-300 pg of synaptosomal protein. Nonspecific uptake was determined in the presence of 10 PM nomifensine (17). After incubation of samples for 5 minutes at 37”C, the reaction was terminated by the addition of 2 ml of ice-cold Krebs buffer and rapid filtration through Whatman GF/F filters. Filters were washed twice with 3 ml of Krebs buffer. The radioactivity associated with the filters was determined using Ecoscint scintillation fluor (National Diagnostics, Manville, NJ). Fluorescence
Polarization
Fluorescence polarization of SPM from rat brain was determined using the fluorescent probe, 1,6-diphenyl-1,3,5hexatriene (DPH). DPH reportedly assesses fluidity at membrane depths corresponding to that of fatty acid sidechains and between lipid bilayers (20). SPM (50 pg protein) were incubated with 1 PM DPH in phosphate-buffered saline, pH 7.4 (136.9 mM NaCl, 2.68 mM KCl, 8.1 mM N&HPO,, 1.47 mM KH,PO,) at 37°C for 30 minutes. Fluorescence was determined in a Perkin Elmer MPF-44B fluorescence spectrophotometer, equipped with rotating polarizing filters. Excitation and emission wavelengths of 360 and 430, respectively, were used (32). Polarization (P) was calculated directly from equation 1 which gives the formula for the Pcorrectedvalue.
CHOLESTEROL
EFFECTS
ON DOPAMINE
UPTAKE
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CHOLESTEROL/PHOSPHOLlPlD 10
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20
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(HINUTES)
FIG. 1. Cholesterol transfer by lipid transfer protein. Cholesterol transfer activity was assessed by measuring the transfer of [“Clcholesterol from liposomes to heat-treated mitochondria in the presence of the lipid transfer protein. [sH]-triolein was included in the liposomes as a nontransferrable marker. Nonspecific transfer of radioactivity was determined in the absence of the lipid transfer protein. Nonspecific transfer was typically less than 20% of the total transfer.
P=
Ivv - Ivh (Ihv/Ihh) (1)
Ivv + Ivh (IhvlIhh)
The term I refers to intensity; h and v indicate the position (horizontal or vertical) of the excitation and emission polarizers. Over a lo-fold range of SPM (10-100 pg protein) P values increased 5% and r values [r=2P/(3-P)] increased 6%. These findings suggest there was minimal nontrivial scatter (11). RESULTS
A fraction containing the lipid transfer protein was isolated from beef liver. The specific activity of this fraction (as defined by its ability to transfer cholesterol from liposomes to mitochondria) was 217 times that of the 5.1 supematant. A major protein in this fraction had a molecular weight of - 14.3 kd, similar to that reported for a lipid transfer protein which is known to transfer cholesterol. [Molecular weights of transfer proteins are reviewed in (40)]. The transfer protein facilitated the net transfer of cholesterol to and from synaptosomes and SPM without producing a marked change in the synaptic phospholipid concentration. The lipid transfer protein facilitated the transfer of cholesterol in a linear manner for at least 25 minutes (Fig. 1). Nonspecific transfer accounted for ~20% of the total cholesterol transferred. Typical cholesterol/phospholipid ratios of unmodified synaptosomes and SPM from rat were 0.44kO.04 and 0.54+0.03, respectively. The value for synaptosomes is lower than that of SPM because the synaptosomal fraction contains mitochondria, which are cholesterol-poor. Figure 2 depicts data obtained from 4 experiments, which each included control, cholesterol-loaded and cholesterol-
FIG. 2. Fluorescence polarization of DPH in rat brain SPM as a function of the cholesterol/phospholipid ratio. The fluorescence polarization of DPH was determined at 37°C. Each symbol in the value (equation 1) obtained from individual inset depicts the Pcorreeted SPM samples, with the indicated cholesterol/phospholipid ratios. The major tIRure represents a compilation of data from four experiments, which included control and cholesterol-modified SPM. Each Pcorrectedvalue is expressed as the % of the control value (unmodified SPM). The values for the four unmodified SPM are Riven as lOC%. P values for control SPM were 0.297kO.026. P values for cholesterolloaded SPM were significantly greater than those for cholesteroldepleted SPM @
depleted SPM. At 37°C P values for control SPM (cholesterol/ phospholipid ratios of 0.54+0.03) were 0.297kO.026. The polarization data from these experiments has been depicted as % of control values to emphasize the relative changes in P which accompany alterations in the synaptic cholesterol/ phospholipid ratio. In each of the 4 experiments, P values for cholesterol-loaded SPM were significantly greater than those for cholesterol-depleted SPM. Representative P values are provided in the inset to Fig. 2. Raising the cholesteroYphospholipid ratio by 10 or more percent increased P by -20%; lowering the ratio by 10% decreased P by -18%. The P values at the cholesterol/ phospholipid ratios +/-lo% of control values appear to be near, respectively, the PpLateau(maximal P) and P,,,, (P value at zero cholesterol) values for SPM (39) since greater changes in the ratios (beyond 10%) produced little further consistent changes in polarization. However, polarization measurements of cholesterol-modified SPM may underestimate changes in membrane fluidity since there is a small but significant change in DPH lifetime in SPM (16), during a developmental period which corresponds to marked change in cholesterol/phospholipid ratios (15). Figure 3A depicts a typical experiment in which the uptake of PHI-dopamine by unmodified (control) synaptosomes was assessed. Nonspecific uptake, determined in the presence of 10 PM nomifensine, accounted for 15-2% of the total filter-associated radioactivity. The K,,, and V,,,,, were calculated from the X-intercept and the slope of the Hanes plot, respectively (Fig. 3B). The K, for dopamine uptake by unmodified synaptosomes was typically 136 nM, while the Vmax was 8.81 pmole/mg protein/5 minutes. The relationship of the cholesterol/phospholipid ratio to
MAGUIRE
AND DRUSE
600
300
900
ml DOPMINE
.2
FIG. 3. Dopamine uptake by control synaptosomes. (A) depicts a typical experiment in which the uptake of dopamine into control synaptosomes was assessed using 10-15 nM PHI-dopamine and 7 concentrations of unlabelled dopamine ranging from O-1000 nM. Each point represents the mean value obtained from triplicate samples. Nonspecific uptake, determined in .the presence of 10 PM nomifensine, accounted for 15-2% of the total uptake. (B) depicts the Hanes plot (substrate concentration/velocity versus substrate concentration) which was used to calculate the K, (136 nM) and V,,, (8.8 pmole/mg protein15 minutes assay).
the K,,, and V,,,,, for synaptosomal uptake of dopamine is illustrated in Figs. 4 and 5. As shown, cholesterol/ phospholipid ratios below the control value for synaptosomes (0.44) had little influence on either the K, or the V,,,,, In contrast, elevated cholesterohphospholipid ratios resulted in a marked decrease in affinity (increased K,,,) and a large increase in the maximum velocity (increased V,,,). K,,, and Vmax values were more than 2 standard deviations above normal when cholesterol/phospholipid ratios were, respectively, -3% or -4% above normal. In fact, a 3&U% increase in the cholesteroYphospholipid ratio resulted in a 3- to 6-fold increase in K,,, and a 4-fold increase in V,,, . A 30-m increase in the cholesterol/phospholipid ratio corresponds to an -2% increase in P (Fig. 2). By comparison, a 30-4% decrease in the SPM cholesterol/phospholipid ratio corresponds to an -18% decrease in P (Fig. 5) a small (2%) decrease in V,,,,, (Fig. 5). and little change in K, (Fig. 4). DISCUSSION
The present study examined the influence of the synaptic cholesterol/phospholipid ratio on membrane fluidity and dopamine uptake. Utilization of a lipid transfer protein facilitated the rapid modification of the synaptic cholesterol! phospholipid ratio over a wide range. We were able to modify the cholesterol/phospholipid ratio of SPM from 0.54 to values ranging from -0.32 to -0.90 and that of synaptosomes from 0.44 to values ranging from -0.30 to - 1.10. According to our interpretation of data from other laboratories, the cholesterol/phospholipid ratio of synaptic membranes from very young animals may be less than half the values of young-mature animals (15). Values from aged animals may be
.6
.4
.0
1.0
1.2
CHOLESTEROL/PHOSPHOLlPlD
(NM)
FIG. 4. The K, for dopamine uptake as a function of the cholesteroYphospholipid ratio. Dopamine uptake was assessed in cholesterol-depleted, control, and cholesterol-loaded synaptosomes. Each point represents the K, obtained from individual uptake assays, such as that described in Fig. 3. Each of the 25 or more assays was performed on a different sample of synaptosomes. Only assays with a correlation coefficient of 0.9 or better for the linear fit of the Hanes plot were included. The error bar represents the meanrstandard deviation of values obtained from control synaptosomes. having a cholesterol’phospholipid ratio of 0.44.
30 25 Y 5
20
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15
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10
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5 0 .2
.4
.6
.8
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1.2
CHOLESTEROL/PliOSPHOLIPID
FIG. 5. The V,,, for dopamine uptake as a function of the cholesteroUphospholipid ratio. Dopamine uptake was assessed in cholesterol-depleted, control, and cholesterol-loaded synaptosomes. Each point represents the V,,,,, obtained from individual uptake assays, such as that described in Fig. 3. Each of the 25 or more assays was performed on a different sample of synaptosomes. Only assays with a correlation coefficient of 0.9 or better for the linear fit of the Hanes plot were included. The error bar represents the mean-cstandard deviation of values obtained from control synaptosomes. having a cholesteroUphospholipid ratio of 0.44.
CHOLESTEROL
EFFECTS
ON DOPAMINE
UPTAKE
more than 40% above those from young-mature animals (33). Another condition which reportedly results in increased cholesteroYphospholipid ratios in synaptic membranes is chronic exposure to alcohol [examples given in (6,3.5)]. Chronic exposure to ethanol reportedly can lead to a 20-30% increase in the cholesterollphospholipid ratio of rodents. Since the numbers presented here may represent the extremes of values reported in the literature, a more conservative estimate of the physiological range of cholesterol/phospholipid ratios would be +3@% of the values obtained in young-mature rats. In our studies, synaptosomes having cholesterol/phospholipid ratios *3(rr of the control values would have ratios of 0.31 to 0.57, while SPM would have ratios from 0.38 to 0.70. Alterations in the cholesterohphospholipid ratio were accompanied by changes in dopamine uptake and by changes in fluorescence polarization of DPH. Although the range of the cholesterol/phospholipid ratios obtained in modified membranes exceeds that expected to accompany development and aging or chronic alcohol consumption, significant changes were also found within the presumed physiological range of cholesterol/phospholipid ratios (?30% of control values). The present studies also demonstrated that increasing the cholesterol/phospholipid ratio markedly affects dopamine uptake. For example, a 30% increase in the cholesterol/ phospholipid ratio produced a 3-fold increase in the K, for dopamine uptake. This change in cholesterollphospholipid ratio was accompanied by a 24% increase in the fluorescence polarization of DPH. Although an increased cholesterol/phospholipid ratio was also associated with an increase in the V,,,,, for dopamine uptake, a higher concentration of dopamine would be required to reach the maximum velocity because of the increased K,. In fact, at concentrations of dopamine below the K,, the rate of dopamine uptake would be decreased because the first order rate constant at low substrate concentrations (3). the V,,,,,/K,,, , was decreased at elevated cholesterol/phospholipid ratios. Thus, the increase in V,,, was proportionately smaller than the increase in &. Since synaptic uptake of dopamine must, be definition, involve a transmembrane process, it is not surprising that this process would be sensitive to changes in the membrane environment of the transporter. If the dopamine uptake transporter was normally located in a phospholipid-rich, cholesterol-poor region of the membrane, increases in the cholesterol/phospholipid ratio would significantly alter both the lipid environment of the transporter and membrane order in the region of the transporter. Such changes could have a marked impact on dopamine uptake. In fact, the results of the present studies suggest that the dopamine transporter may normally be found in such a region since it is affected by high cholesterol content, but not by low cholesterol content. This is shown by the fact that decreasing the cholesterol phospholipid ratio (and fluorescence polarization) from the control value had little effect on the transporter, while increasing the cholesterol/phospholipid ratio altered both the K, and the V,,,,,. At the present time, it is not possible to determine whether the effects of high cholesterol on the dopamine transporter are due solely to an increase in membrane order, to an increase in the cholesterol content in the region of the transporter, or to a combination of these two possibilities. A kinetic analysis of a simple mechanism for dopamine uptake suggests that one step affected by the increased cholesteroYphospholipid ratio is beyond the initial binding of
435
dopamine to the transporter, T. In order for dopamine to be transported across the synaptic membrane, dopamine must bind to the transporter, T, on the synaptic cleft side of the membrane. In addition, dopamine must be released inside of the synapse. This simple, hypothetical model for dopamine uptake and transport into a cell can be defined in kinetic terms by the equation below. This equation describes the initial rate of dopamine uptake under the experimental conditions where the concentration of radiolabeled dopamine outside of the synaptosome is much greater than that within the synaptosome. k, kf T.dopamine 5 T + dopaminq, I The K, and V,,,,, will be defined as: k2 + k-, K, = k, T + dopamine,,,
(2)
Vma,= k,[‘f’l Consider the observation that an increase in cholesterol/ phospholipid ratios was accompanied by an increase in both the K, and the V,,,,,. Since the concentration of transporter, (T), was held constant in these in vitro assays, the observed increase in Vmax may be explained by an increase in k2. An increase in kZ would also result in an increase in K,,, under conditions where k_, is not much greater than kz. Thus, it appears likely that the increased synaptic cholesterol/ phospholipid ratio influenced dopamine transport at the level of k2. which is a complex constant, presumably reflecting the contributions of a number of steps which follow the initial binding of dopamine to the transporter. Alterations in the synaptic content of cholesterol also appears to affect other transmembrane processes. It has been shown that incorporation of cholesterol by hippocampal slices abolishes carbachol-stimulation of pyramidal cells in the CA1 region (7). In addition, work in this laboratory suggests that elevated synaptic cholesterol/phospholipid ratios result in decreased stimulation of striatal adenylate cyclase by dopamine (10). However, it is not possible to generalize the findings reported here to all transmembrane processes. In fact, it appears that alteration of the synaptic cholesterol/phospholipid ratio has the opposite effect on the uptake of gammaaminobutyric acid (GABA). Whereas GABA uptake appears to be insensitive to increased cholesterol/phospholipid ratios, it is inhibited by decreased cholesteroVphospholipid ratios (27). The difference in the sensitivities of the dopamine and GABA transporters may reflect differences in the nature of the membrane environment in which the transporters are normally found or in the lipid requirements of the transporters. Thus, transmembrane processes may be selectively sensitive to changes in the lipid composition and fluidity of their membranous environments. The results of the present studies have demonstrated that the transmembrane process of dopamine uptake may be inhibited by elevated synaptic cholesterol/phospholipid ratios, which are accompanied by increased fluorescence polarization. These results may be relevant to changes which occur during aging since there are reports of increased synaptic cholesterohphospholipid ratios and decreased membrane fluidity in aged animals (1, 13, 15, 26, 31). Unless compensatory changes in membrane composition accompany the development of the age-related increase in synaptic cholesterol, one might expect to find decreased dopamine uptake in aged animals. In fact, synaptosomal uptake of
MAGUIRE
436
dopamine is reportedly decreased in senescent versus mature mice (18). However, compensatory membrane changes may occur in rats since others have found no significant differences in dopamine uptake in aged rats (24,36).
AND DRUSE
Nonetheless, in light of the evidence of an age-related loss of striatal dopamine and dopaminergic receptors [reviewed in (19)], a cholesterol-induced decrease in dopamine uptake could potentially worsen dopaminergic function in the aged.
ACKNOWLEDGEMENTS
The authors would like to thank Dr. Allen Frankfater for the assistance he provided in the kinetic and fluorescence analyses and interpretation of the data.
REFERENCES 1. Bonetti, A. C.; Battistella, A.; Calderini, G.; Teolato, S.; Crews, F. T.; Gaiti, A.; Algeri, S.; Toffano, G. Biochemical alterations in the mechanisms of synaptic transmission in aging - brain. In: Samuel, D.; Algeri, S.;.Gershon, S.; Grimm, V. E.; Toffano, G.. eds. Aaina of the brain. New York: Raven Press: 1983:17i-18i. - 2. Chweh, A. Y.; Leslie, S. W. Phosphatidylserine enhancement of PHI-gamma-aminobutyric acid uptake by rat whole brain synaptosomes. J. Neurochem. 38691-695; 1982. 3. Comish-Bowden, A. Fundamentals of enzyme kinetics. Boston: Butterworth, Inc.; 1979:20-24. 4. Cotman, C.; Matthews, D. Synaptic plasma membranes from rat brain synaptosomes: isolation and partial characterization. Biochim. Biophys. Acta 249:38&394; 1971. 5. Crain, R. C.; Zilversmit, D. B. Two nonspecific phospholipid exchange proteins from beef liver. I. Purification and characterization. Biochemistry 19: 1433-1439; 1980. 6. Crews, F. T.; Majchrowicz, E.; Meeks, R. Changes in cortical synaptosomal plasma membrane fluidity and composition in ethanol-dependent rats. Psychopharmacology (Berlin) 81:201 213; 1983. 7. Crews, F. T.; &macho, A.; Philips, M. I. Cholinergic stimulation of hippocampal pyramidal cells is inhibited by increasing membrane cholesterol. Brain Res. 261: 155-158; 1983. 8. DeBlasi, A.; Mennini, T. Selective reduction of one class of dopamine receptor binding sites in the corpus striatum of aged rats. Brain Res. 242:361-364; 1982. 9. Deskin, R.; Seidler, F. J.; Whitmore, W. L.; Slotkin, T. A. Development of cx-noradrenergic and dopaminergic receptor systems depends upon maturation of their presynaptic nerve terminals in the rat brain. J. Neurochem. 36:1683-1690; 1981. 10. Druse, M. J.; Maguire, P. A. Cholesterol effects on Dl binding & dopamine-stimulated adenylate cyclase. Trans. Am. Sot. Neurochem. 19:184; 1988. 11. Eisinger, J.; Flares, J. Fluorometry of turbid and absorbant samples and the membrane fluidity of intact erythrocytes. Biophys. J. 4877-84; 1985. 12. Fiske, C. H.; Subbarow, Y. The calorimetric determination of phosphorus. J. Biol. Chem. 66:375-400: 1925. 13. Freuhd, G. The interaction of chronic alcohol consumption and aging on brain structure and function. Alcoholism (NY) 6: 13-21; 1982. 14. Heron, D. S.; Shinitzky, M.; Hershkowitz, M.; Samuel, D. Lipid fluidity markedly modulates the binding of serotonin to mouse brain membranes. Proc. Natl. Acad. Sci. USA 12:74637467; 1980. 15. Hitzemann, R. J.; Johnson, D. A. Developmental changes in synaptic membrane lipid composition and fluidity. Neurochem. Res.-8:121-131; 1983._ 16. Hitzemann, R. J.; Harris, R. A. Developmental changes in synaptic membrane fluidity: a comparison of 1,6-diphenyl1,3,5-hexatriene (DPH) and 1-[4-(trimethylamino)phenyl]-6phenyl-1,3,5-hexatriene (TMA-DPH) Dev. Brain Res. 14:113120; 1984.
17. Hunt, P.; Kannengiesser, M.; Raynaud, J. Nomifensine: a new potent inhibitor of dopamine uptake into synaptosomes from rat brain corpus striatum. J. Pharm. Pharrnacol. 26:370-371; 1974. 18. Jonec, V.; Finch, C. E. Ageing and dopamine uptake by subcellular fractions of the C57BW6J male mouse brain. Brain Res. 91:197-215; 1975. 19. Joseph, J. A.; Roth, G. S. Age-related alterations in dopaminergic mechanisms. In: Samuels, D.; Algeri, S.; Gershon, S.; Grimm, V. E.; Toffano, G., eds. Aging of the brain. New York: Raven Press; 1983:245-256. 20. Jost, P. C.; Griffhh, 0. H.; Lipid-lipid and lipid-protein interactions in membranes. Pharmacol. Biochem. Behav. 13(Suppl. 1):155-165: 1980. 21. Lowry, 0. H.; Rosebrough, N. H.; Farr, A. L.; Randall, R. J. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193:265-275; 1951. 22. Makman, M .H.; Ahn, H. S.; Thal, L. J.; Sharpless, B.; Dvorkin, B.; Horowitz, S. G.; Rosenfeld, M. Evidence for selective loss of brain dopamine- and histamine-stimulated adenylate cyclase activities in rabbits with aging. Brain Res. 192:177-183; 1980. 23. Marshall, J. F.: Altar. C. A. Striatal donamine untake and swim performance in the aged rat. Brain Res’. 379: 1Ii-1 17; 1986. 24. Memo, M.; Lucchi, L.; Spano, P. F.; Trabucchi, M. Aging process affects a single class of dopamine receptors. Brain Res. 202488-492; 1980. 25. Misra, C. H.; Shelat, H. S.; Smith, R. C. Effect of age on adrenergic and dopaminergic receptor binding in rat brain. Life Sci. 27:521-526; 1980. 26. Nagy, K.; Simon, P.: Zs.-Nagy, I. Spin label studies on synaptosomal membranes of rat brain cortex during aging. Biochem. Biophys. Res. Commun. 117:688-694; 1983. 27. North, P.; Fleischer, S. Alteration of synaptic membrane cholesterohphospholipid ratio using a lipid transfer protein. Effect on gamma-aminobutyric acid uptake. J. Biol. Chem. 258: 12421253; 1983. 28. North, P.; Fleischer, S. Use of a nonspecific lipid transfer protein to modify the cholesterol content of synaptic membranes. Methods Enzymol. 98:599-613; 1983. 29. Pagano, R. E.; Ozato, K.: Ruysschaert, J. M. Intracellular distribution of lipophilic fluorescent probes in mammalian cells. Biochim. Biophys. Acta 465661-666; 1977. 30. Roth, G. S. Altered mechanisms of opiate and dopaminergic action during aging. In: Giacobini, E.; Filogamo, G.;‘Giacobini, G.; Vemadakis, A., eds. The aaina brain: Cellular and molecular mechanisms of aging in td nervous system. New York: Raven Press; 1982:203-209. 31. Samuel, D.; Heron, D. S.; Hershkowitz, M.; Shinitzky, M. Aging receptor binding and membrane microviscosity. In: Giacobini, E.; Filogamo, G.; Giacobini, G.; Vemadakis, A., eds. The aging brain: cellular and molecular mechanisms of aging in the nervous system. New York: Raven Press; 1982:9397.
CHOLESTEROL
EFFECTS ON DOPAMINE
UPTAKE
32. Shinitzky, M.; Barenholz, Y. Fluidity parameters of lipid regions determined by fluorescence polarization. Biochim. Biophys. Acta 515:367-394; 1978. 33. Shinitzky, M.; Heron, D. S.; Samuel, D. Restoration of membrane fluidity and serotonin receptors in the aged mouse brain. In: Samuel, D.; Algeri, S.; Gershon, S.; Grimm, V. E.; Toffano, G., eds. Aging of the brain. New York: Raven Press; 1983:32% 336. ___. 34. Sklar, L. A. Fluorescence polarization studies of membrane fluiditv: where do we PO from here? In: Kates. M.: Manson. L. A., eds. Membrane- fluidity. New York:’ Plenum Press; 1984:9%131. 35. Smith, T. L.; Gerhart, M. J. Alterations in brain lipid composition of mice made physically dependent to ethanol. Life Sci. 31:141%1425; 1982. 36. Strong, R.; Samorajski, T.; Gottesfeld, Z. High affinity uptake of neurotransmitters in rat neostriatum: effects of aging. J. Neurochem. 43:1766-1768; 1984.
437
37. Sun, G. Y.; Sun, A. Y. Chronic ethanol administration induced an increase in phosphatidylserine in guinea pig synaptic plasma membranes. Biochem. Biophys. Res. Commun. 113:262-268; _ 1983. 38. Trabucchi, M.; Spano, P. R.; Govoni, S.; Riccardi, F.; Bosio, A. Dopaminergic function during aging in rat brain. In: Giacobini, E.; Filogamo, G.; Giacobini, G.; Vemadakis, A., eds. The aging brain: Cellular and molecular mechanisms of aging in the nervous system. New York: Raven Press; 1982:195-201. 39. van Blitterswijk, W. J.; van der Meer, B. W.; Hilkmann, J. Quantitative contributions of cholesterol and the individual classes of phospholipids and their degree of fatty acyl (un) saturation to membrane fluidity measured by fluorescence polarization. Biochemistry 26: 1746-1756; 1987. 40. Zilversmit, D. Lipid transfer proteins: overview and applications. Methods Enzymol. 98:565-573; 1983.
0 Pergamon Press plc, 1989. Printed in the U.S.A.
0361-9230/89 $3.00 + .OO
The Influence of Cholesterol on Synaptic Fluidity and Dopamine Uptake’ PATRICIA
A. MAGUIRE2
AND MARY
J. DRUSE3
Department of Biochemistry and Biophysics Loyola University Stritch School of Medicine, Maywood, Received MAGUIRE,
P. A. AND M. J. DRUSE.
The influence
1 July
of cholesterol
IL 601.53
1988 on
synapticfluidity
and dopamine
uptake.
BRAIN
RES BULL 22(2) 431-437, 1989.-The present study examined how the synaptic cholesteroVphospholipid ratio influences membrane fluidity and a transmembrane process of the dopaminergic system, dopamine uptake. Fluorescence polarization of DPH was used as a measure of membrane fluidity. The cholesterol content of synaptosomal and synaptic plasma membranes was altered using a lipid transfer protein. The results of the present study demonstrate that the transmembrane process of dopamine uptake may be inhibited by an elevated synaptic cholesterol/phospholipid ratio, which correlates with increased fluorescence polarization. Specifically, a l&30% increase in the cholesterol/phospholipid ratio resulted in an -20% increase in fluorescence polarization, a 3- to 6-fold increase in K,, a 4-fold increase in V,,, , and a marked decrease in the V,,,,,/K,,, (the first order rate constant for dopamine uptake at low substrate concentrations). Fluidity
Dopamine
Uptake
Synaptic plasma membranes
THERE is evidence that as an animal matures and ages, the cholesteroYphospholipid ratio of synaptic membranes increases and membrane fluidity decreases (1, 13, 15, 26, 31). These synaptic membrane changes are accompanied, temporally, by alterations in the components of several neurotransmitter systems [reviewed in (30)]. Since many membrane-associated functions are markedly influenced by membrane lipids and fluidity [examples given in (2, 27, 28, 31, 37)], it is possible that at least some of the age-associated changes of the neurotransmitter systems are related to alterations in the cholesterol/phospholipid ratio and fluidity of synaptic membranes. In an attempt to investigate this hypothesis, we examined the influence of the synaptic cholesterol/phospholipid ratio on the fluorescence polarization of l,ddiphenyl-1,3,5-hexatriene (DPH) and on a membrane-associated function of the dopaminergic system. The dopaminergic system was studied because several components of this system appear to be altered by aging (8, 18, 22, 24, 25, 38). Specifically, we examined the influence of the cholesterol/ phospholipid ratio on the uptake of dopamine. Dopamine uptake was studied since such a transmembrane process might be expected to be sensitive to changes in membrane composition and fluidity. The cholesterol content was altered using a lipid transfer protein (28) which facilitates the rapid transport of cholesterol between synaptic membranes and liposomes.
Synaptosomes
Cholesterol
METHOD Preparation
of Liposomes
Three types of liposomes were used in transfer experiments. One type of liposome was used to determine the activity of the lipid transfer protein by assessing the ability of the protein to transfer cholesterol from liposomes to mitochondria. These liposomes were prepared using 1 mg phosphatidylcholine (PC) per ml and 1 mole cholesterol per mole of PC and contained 0.25 @Zi each of [WI-cholesterol (Amersham, Arlington Heights, IL) and PHI-triolein (ICN Radiochemicals, Irvine, CA) as transferrable and nontransferrable markers, respectively (28). The other two types of liposomes were used in experiments in which synaptosomes and synaptic plasma membranes @PM) were either loaded with or depleted of cholesterol. That is, they were used in experiments in which the lipid transfer protein facilitated a net transfer of cholesterol from liposomes to synaptosomes or SPM (cholesterol loading) or from synaptosomes or SPM to liposomes (cholesterol depletion). For cholesterol depletion studies, liposomes were prepared using 10 mg egg PC per ml. Those to be used for cholesterol loading were prepared with 5 mg PC/ml and a cholesterol/ phospholipid molar ratio of - 1.7. Both of the latter types of liposomes also contained 0.25 &i of the nontransferrable marker [“‘Cl-tripalmitin (Amersham, Arlington Height, IL). To prepare liposomes, lipids were mixed in chloroform and
‘This research was submitted in partial fulfillment of the Ph.D. requirements of Loyola University of Chicago (P.A.M.). *Present address: Department of Biology, University of California, Riverside, CA 92521. 3Requests for reprints should be addressed to Mary J. Druse Manteuffel.
431
MAGUIRE dried, under a stream of nitrogen, to a thin film on the sides of an acid-washed tube. Lipid films were dispersed in 0.32 M sucrose, 5 mM Tris, pH 7.0, by extensive mixing on a Vortex mixer. The mixed samples were then sonicated until the lipid mixture was translucent. Sonicated liposomes were centrifuged at 27,000 g for 60 minutes and used within 3 hours of preparation. isolation of Lipid Transfer Protein
The nonspecific lipid transfer protein was partially purified from beef liver, obtained from Aurora Packing Company (North Aurora, IL), by a modification of previous procedures of (5,28). All procedures were done at 4°C. A 3% (w/v) homogenate was prepared in a buffer containing 0.25 M sucrose, 1 mM EDTA, 50 mM Tris-HCl, and 0.0% (w/v) sodium azide, pH 7.4. The homogenate was centrifuged at 13,000 g for 30 minutes. The pH of the supematant was adjusted to 5.1 with HCl and mixed for 1 hour prior to centrifugation at 13,000 g for 20 minutes. The pH of the clear supematant (5.1 supematant) was returned to 7.4 with NaOH. Aliquots were stored at -20°C for up to 6 months. Protein in the 5.1 supematant was precipitated with ammonium sulfate between 4% and 90% saturation. The resulting pellet was suspended in a buffer containing 25 mM sodium phosphate, 10 mM 2-mercaptoethanol and 0.0% (w/v) sodium azide, pH 8.1, and dialyzed extensively against a solution containing 5 mM sodium phosphate, 5 mM 2mercaptoethanol, 0.0% (w/v) sodium azide, pH 8.1. The dialysate was applied to a Sephadex CM-C25 (Pharmacia, Inc., Uppsala, Sweden) ion exchange column and washed extensively with dialysis buffer. Transfer activity was eluted with a solution containing 25 mM sodium phosphate, 45 mM NaCl, 5 mM 2-mercaptoethanol and 0.0% (w/v) sodium azide, pH 8.1 (elution buffer). Transfer activity was stable in elution buffer for 1 month at 4°C. Prior to use, the transfer protein was dialyzed against the elution buffer without azide and concentrated 5- to IO-fold by ultrafiltration through an Amicon cell, equipped with a Diaflo Y-M5 membrane (Amicon Corp., Danvers, MA). The ability of the purified proteins to transfer cholesterol between liposomes and membranes was assessed by the standard procedure (28) which uses beef heart mitochondria (4) as the cholesterol acceptor. Mitochondria are typically used as an acceptor because of their low cholesterol content and their ease of preparation. Isolation of Synaptosomes Membranes (SPM)
and Synaptic Plasma
Synaptosomes and SPM were isolated from the brains of 40-day-old male Sprague-Dawley rats (Holtzman, Madison, WI) (4) and used immediately thereafter in cholesterol loading and depletion experiments. Normal and lipid-modified synaptosomes were used to assess dopamine uptake. However, SPM were used in fluorescence polarization studies rather than synaptosomes in order to minimize the partitioning of the hydrophobic fluorescent probes into membranes other than the synaptic plasma membrane (e.g., synaptosomal mitochondria and vesicles). The importance of using purified plasma membranes as a means of minimizing membrane heterogeneity has been stressed previously (29,34). Although synaptosomal and SPM preparations are not identical, one should be able to cautiously extrapolate the results from one to the other. Aliquots of control and modified synaptosomes and SPM
AND DRUSE
were stored at -80°C prior to analysis of protein (21), phospholipid (12), and cholesterol (Sigma Kit 351, Sigma, St. Louis, MO). Fifty micrograms of SPM protein were used routinely in the fluorescence polarization studies. Fifty micrograms of SPM protein corresponds to -37 pg phospholipid and -10 ,ug cholesterol in control SPM. In a comparable sample of modified SPM the phospholipid content was not significantly altered, however, the cholesterol content ranged from -6.5 to -16.7 pg. Two to three hundred micrograms of synaptosomal protein were used in dopamine uptake experiments. Lipid Transfer Between Liposomes
and Synaptosomes
Synaptosomes or SPM (1 mg protein in 50 ~1 buffered 0.32 M sucrose) were preincubated for 10 minutes at 32°C with 200 ~1 of concentrated transfer protein in elution buffer, 80 ~1 of 1.8 M sucrose, and 170 ~1 of deionized water (final sucrose concentration = 0.32 M). The 200 ~1 of concentrated transfer protein contained from 0.1 to 0.4 mg of protein. The reaction was initiated by the addition of 500 ~1 of liposomes, and terminated after either 30 (synaptosomes) or 60 minutes @PM) by centrifugation at 106,000 g for 40 minutes through a layer 0.5 M sucrose (28). Pellets were rinsed and suspended in the appropriate buffer for subsequent analyses. Control and modified synaptosomes were gently suspended in modified Krebs-Henseleit buffer (118 mM NaCl, 26 mM NqHPQ, 6 mM NaH,P04, 4.7 mM KCl, 1.8 CaCl,, 1.2 mM MgS04, 1.3 mM EDTA, 5.6 mM D-glucose, 2 FM ascorbic acid, 10 PM pargyline, pH 7.4) while SPM were suspended in a solution containing 0.32 M sucrose and 5 mM Tris, pH 7.0. Dopamine
Uptake
Synaptosomal uptake of dopamine was assayed by a modification of a standard procedure (9). Triplicate tubes were prepared which each contained 10-15 nM rH]dopamine (New England Nuclear, Boston, MA) and O-5000 nM unlabeled dopamine in 900 ~1 of the Krebs buffer. The reaction was initiated by the addition of 100 ~1 aliquots containing 200-300 pg of synaptosomal protein. Nonspecific uptake was determined in the presence of 10 PM nomifensine (17). After incubation of samples for 5 minutes at 37”C, the reaction was terminated by the addition of 2 ml of ice-cold Krebs buffer and rapid filtration through Whatman GF/F filters. Filters were washed twice with 3 ml of Krebs buffer. The radioactivity associated with the filters was determined using Ecoscint scintillation fluor (National Diagnostics, Manville, NJ). Fluorescence
Polarization
Fluorescence polarization of SPM from rat brain was determined using the fluorescent probe, 1,6-diphenyl-1,3,5hexatriene (DPH). DPH reportedly assesses fluidity at membrane depths corresponding to that of fatty acid sidechains and between lipid bilayers (20). SPM (50 pg protein) were incubated with 1 PM DPH in phosphate-buffered saline, pH 7.4 (136.9 mM NaCl, 2.68 mM KCl, 8.1 mM N&HPO,, 1.47 mM KH,PO,) at 37°C for 30 minutes. Fluorescence was determined in a Perkin Elmer MPF-44B fluorescence spectrophotometer, equipped with rotating polarizing filters. Excitation and emission wavelengths of 360 and 430, respectively, were used (32). Polarization (P) was calculated directly from equation 1 which gives the formula for the Pcorrectedvalue.
CHOLESTEROL
EFFECTS
ON DOPAMINE
UPTAKE
433 125T
0’
60.
oz
0 0
s
0 100
owooo
0 75 0.300
Oao
0
0
(IDO
: a
NONSPECIFIC
0
0
i
5
w
0
00
O
““,
il
0 0.600
C”ME3lmO”P”mP”oUPID I 0.900
CHOLESTEROL/PHOSPHOLlPlD 10
5 ~tnE
15
20
25
(HINUTES)
FIG. 1. Cholesterol transfer by lipid transfer protein. Cholesterol transfer activity was assessed by measuring the transfer of [“Clcholesterol from liposomes to heat-treated mitochondria in the presence of the lipid transfer protein. [sH]-triolein was included in the liposomes as a nontransferrable marker. Nonspecific transfer of radioactivity was determined in the absence of the lipid transfer protein. Nonspecific transfer was typically less than 20% of the total transfer.
P=
Ivv - Ivh (Ihv/Ihh) (1)
Ivv + Ivh (IhvlIhh)
The term I refers to intensity; h and v indicate the position (horizontal or vertical) of the excitation and emission polarizers. Over a lo-fold range of SPM (10-100 pg protein) P values increased 5% and r values [r=2P/(3-P)] increased 6%. These findings suggest there was minimal nontrivial scatter (11). RESULTS
A fraction containing the lipid transfer protein was isolated from beef liver. The specific activity of this fraction (as defined by its ability to transfer cholesterol from liposomes to mitochondria) was 217 times that of the 5.1 supematant. A major protein in this fraction had a molecular weight of - 14.3 kd, similar to that reported for a lipid transfer protein which is known to transfer cholesterol. [Molecular weights of transfer proteins are reviewed in (40)]. The transfer protein facilitated the net transfer of cholesterol to and from synaptosomes and SPM without producing a marked change in the synaptic phospholipid concentration. The lipid transfer protein facilitated the transfer of cholesterol in a linear manner for at least 25 minutes (Fig. 1). Nonspecific transfer accounted for ~20% of the total cholesterol transferred. Typical cholesterol/phospholipid ratios of unmodified synaptosomes and SPM from rat were 0.44kO.04 and 0.54+0.03, respectively. The value for synaptosomes is lower than that of SPM because the synaptosomal fraction contains mitochondria, which are cholesterol-poor. Figure 2 depicts data obtained from 4 experiments, which each included control, cholesterol-loaded and cholesterol-
FIG. 2. Fluorescence polarization of DPH in rat brain SPM as a function of the cholesterol/phospholipid ratio. The fluorescence polarization of DPH was determined at 37°C. Each symbol in the value (equation 1) obtained from individual inset depicts the Pcorreeted SPM samples, with the indicated cholesterol/phospholipid ratios. The major tIRure represents a compilation of data from four experiments, which included control and cholesterol-modified SPM. Each Pcorrectedvalue is expressed as the % of the control value (unmodified SPM). The values for the four unmodified SPM are Riven as lOC%. P values for control SPM were 0.297kO.026. P values for cholesterolloaded SPM were significantly greater than those for cholesteroldepleted SPM @
depleted SPM. At 37°C P values for control SPM (cholesterol/ phospholipid ratios of 0.54+0.03) were 0.297kO.026. The polarization data from these experiments has been depicted as % of control values to emphasize the relative changes in P which accompany alterations in the synaptic cholesterol/ phospholipid ratio. In each of the 4 experiments, P values for cholesterol-loaded SPM were significantly greater than those for cholesterol-depleted SPM. Representative P values are provided in the inset to Fig. 2. Raising the cholesteroYphospholipid ratio by 10 or more percent increased P by -20%; lowering the ratio by 10% decreased P by -18%. The P values at the cholesterol/ phospholipid ratios +/-lo% of control values appear to be near, respectively, the PpLateau(maximal P) and P,,,, (P value at zero cholesterol) values for SPM (39) since greater changes in the ratios (beyond 10%) produced little further consistent changes in polarization. However, polarization measurements of cholesterol-modified SPM may underestimate changes in membrane fluidity since there is a small but significant change in DPH lifetime in SPM (16), during a developmental period which corresponds to marked change in cholesterol/phospholipid ratios (15). Figure 3A depicts a typical experiment in which the uptake of PHI-dopamine by unmodified (control) synaptosomes was assessed. Nonspecific uptake, determined in the presence of 10 PM nomifensine, accounted for 15-2% of the total filter-associated radioactivity. The K,,, and V,,,,, were calculated from the X-intercept and the slope of the Hanes plot, respectively (Fig. 3B). The K, for dopamine uptake by unmodified synaptosomes was typically 136 nM, while the Vmax was 8.81 pmole/mg protein/5 minutes. The relationship of the cholesterol/phospholipid ratio to
MAGUIRE
AND DRUSE
600
300
900
ml DOPMINE
.2
FIG. 3. Dopamine uptake by control synaptosomes. (A) depicts a typical experiment in which the uptake of dopamine into control synaptosomes was assessed using 10-15 nM PHI-dopamine and 7 concentrations of unlabelled dopamine ranging from O-1000 nM. Each point represents the mean value obtained from triplicate samples. Nonspecific uptake, determined in .the presence of 10 PM nomifensine, accounted for 15-2% of the total uptake. (B) depicts the Hanes plot (substrate concentration/velocity versus substrate concentration) which was used to calculate the K, (136 nM) and V,,, (8.8 pmole/mg protein15 minutes assay).
the K,,, and V,,,,, for synaptosomal uptake of dopamine is illustrated in Figs. 4 and 5. As shown, cholesterol/ phospholipid ratios below the control value for synaptosomes (0.44) had little influence on either the K, or the V,,,,, In contrast, elevated cholesterohphospholipid ratios resulted in a marked decrease in affinity (increased K,,,) and a large increase in the maximum velocity (increased V,,,). K,,, and Vmax values were more than 2 standard deviations above normal when cholesterol/phospholipid ratios were, respectively, -3% or -4% above normal. In fact, a 3&U% increase in the cholesteroYphospholipid ratio resulted in a 3- to 6-fold increase in K,,, and a 4-fold increase in V,,, . A 30-m increase in the cholesterol/phospholipid ratio corresponds to an -2% increase in P (Fig. 2). By comparison, a 30-4% decrease in the SPM cholesterol/phospholipid ratio corresponds to an -18% decrease in P (Fig. 5) a small (2%) decrease in V,,,,, (Fig. 5). and little change in K, (Fig. 4). DISCUSSION
The present study examined the influence of the synaptic cholesterol/phospholipid ratio on membrane fluidity and dopamine uptake. Utilization of a lipid transfer protein facilitated the rapid modification of the synaptic cholesterol! phospholipid ratio over a wide range. We were able to modify the cholesterol/phospholipid ratio of SPM from 0.54 to values ranging from -0.32 to -0.90 and that of synaptosomes from 0.44 to values ranging from -0.30 to - 1.10. According to our interpretation of data from other laboratories, the cholesterol/phospholipid ratio of synaptic membranes from very young animals may be less than half the values of young-mature animals (15). Values from aged animals may be
.6
.4
.0
1.0
1.2
CHOLESTEROL/PHOSPHOLlPlD
(NM)
FIG. 4. The K, for dopamine uptake as a function of the cholesteroYphospholipid ratio. Dopamine uptake was assessed in cholesterol-depleted, control, and cholesterol-loaded synaptosomes. Each point represents the K, obtained from individual uptake assays, such as that described in Fig. 3. Each of the 25 or more assays was performed on a different sample of synaptosomes. Only assays with a correlation coefficient of 0.9 or better for the linear fit of the Hanes plot were included. The error bar represents the meanrstandard deviation of values obtained from control synaptosomes. having a cholesterol’phospholipid ratio of 0.44.
30 25 Y 5
20
,o ;
15
B ::
10
E
5 0 .2
.4
.6
.8
1.0
1.2
CHOLESTEROL/PliOSPHOLIPID
FIG. 5. The V,,, for dopamine uptake as a function of the cholesteroUphospholipid ratio. Dopamine uptake was assessed in cholesterol-depleted, control, and cholesterol-loaded synaptosomes. Each point represents the V,,,,, obtained from individual uptake assays, such as that described in Fig. 3. Each of the 25 or more assays was performed on a different sample of synaptosomes. Only assays with a correlation coefficient of 0.9 or better for the linear fit of the Hanes plot were included. The error bar represents the mean-cstandard deviation of values obtained from control synaptosomes. having a cholesteroUphospholipid ratio of 0.44.
CHOLESTEROL
EFFECTS
ON DOPAMINE
UPTAKE
more than 40% above those from young-mature animals (33). Another condition which reportedly results in increased cholesteroYphospholipid ratios in synaptic membranes is chronic exposure to alcohol [examples given in (6,3.5)]. Chronic exposure to ethanol reportedly can lead to a 20-30% increase in the cholesterollphospholipid ratio of rodents. Since the numbers presented here may represent the extremes of values reported in the literature, a more conservative estimate of the physiological range of cholesterol/phospholipid ratios would be +3@% of the values obtained in young-mature rats. In our studies, synaptosomes having cholesterol/phospholipid ratios *3(rr of the control values would have ratios of 0.31 to 0.57, while SPM would have ratios from 0.38 to 0.70. Alterations in the cholesterohphospholipid ratio were accompanied by changes in dopamine uptake and by changes in fluorescence polarization of DPH. Although the range of the cholesterol/phospholipid ratios obtained in modified membranes exceeds that expected to accompany development and aging or chronic alcohol consumption, significant changes were also found within the presumed physiological range of cholesterol/phospholipid ratios (?30% of control values). The present studies also demonstrated that increasing the cholesterol/phospholipid ratio markedly affects dopamine uptake. For example, a 30% increase in the cholesterol/ phospholipid ratio produced a 3-fold increase in the K, for dopamine uptake. This change in cholesterollphospholipid ratio was accompanied by a 24% increase in the fluorescence polarization of DPH. Although an increased cholesterol/phospholipid ratio was also associated with an increase in the V,,,,, for dopamine uptake, a higher concentration of dopamine would be required to reach the maximum velocity because of the increased K,. In fact, at concentrations of dopamine below the K,, the rate of dopamine uptake would be decreased because the first order rate constant at low substrate concentrations (3). the V,,,,,/K,,, , was decreased at elevated cholesterol/phospholipid ratios. Thus, the increase in V,,, was proportionately smaller than the increase in &. Since synaptic uptake of dopamine must, be definition, involve a transmembrane process, it is not surprising that this process would be sensitive to changes in the membrane environment of the transporter. If the dopamine uptake transporter was normally located in a phospholipid-rich, cholesterol-poor region of the membrane, increases in the cholesterol/phospholipid ratio would significantly alter both the lipid environment of the transporter and membrane order in the region of the transporter. Such changes could have a marked impact on dopamine uptake. In fact, the results of the present studies suggest that the dopamine transporter may normally be found in such a region since it is affected by high cholesterol content, but not by low cholesterol content. This is shown by the fact that decreasing the cholesterol phospholipid ratio (and fluorescence polarization) from the control value had little effect on the transporter, while increasing the cholesterol/phospholipid ratio altered both the K, and the V,,,,,. At the present time, it is not possible to determine whether the effects of high cholesterol on the dopamine transporter are due solely to an increase in membrane order, to an increase in the cholesterol content in the region of the transporter, or to a combination of these two possibilities. A kinetic analysis of a simple mechanism for dopamine uptake suggests that one step affected by the increased cholesteroYphospholipid ratio is beyond the initial binding of
435
dopamine to the transporter, T. In order for dopamine to be transported across the synaptic membrane, dopamine must bind to the transporter, T, on the synaptic cleft side of the membrane. In addition, dopamine must be released inside of the synapse. This simple, hypothetical model for dopamine uptake and transport into a cell can be defined in kinetic terms by the equation below. This equation describes the initial rate of dopamine uptake under the experimental conditions where the concentration of radiolabeled dopamine outside of the synaptosome is much greater than that within the synaptosome. k, kf T.dopamine 5 T + dopaminq, I The K, and V,,,,, will be defined as: k2 + k-, K, = k, T + dopamine,,,
(2)
Vma,= k,[‘f’l Consider the observation that an increase in cholesterol/ phospholipid ratios was accompanied by an increase in both the K, and the V,,,,,. Since the concentration of transporter, (T), was held constant in these in vitro assays, the observed increase in Vmax may be explained by an increase in k2. An increase in kZ would also result in an increase in K,,, under conditions where k_, is not much greater than kz. Thus, it appears likely that the increased synaptic cholesterol/ phospholipid ratio influenced dopamine transport at the level of k2. which is a complex constant, presumably reflecting the contributions of a number of steps which follow the initial binding of dopamine to the transporter. Alterations in the synaptic content of cholesterol also appears to affect other transmembrane processes. It has been shown that incorporation of cholesterol by hippocampal slices abolishes carbachol-stimulation of pyramidal cells in the CA1 region (7). In addition, work in this laboratory suggests that elevated synaptic cholesterol/phospholipid ratios result in decreased stimulation of striatal adenylate cyclase by dopamine (10). However, it is not possible to generalize the findings reported here to all transmembrane processes. In fact, it appears that alteration of the synaptic cholesterol/phospholipid ratio has the opposite effect on the uptake of gammaaminobutyric acid (GABA). Whereas GABA uptake appears to be insensitive to increased cholesterol/phospholipid ratios, it is inhibited by decreased cholesteroVphospholipid ratios (27). The difference in the sensitivities of the dopamine and GABA transporters may reflect differences in the nature of the membrane environment in which the transporters are normally found or in the lipid requirements of the transporters. Thus, transmembrane processes may be selectively sensitive to changes in the lipid composition and fluidity of their membranous environments. The results of the present studies have demonstrated that the transmembrane process of dopamine uptake may be inhibited by elevated synaptic cholesterol/phospholipid ratios, which are accompanied by increased fluorescence polarization. These results may be relevant to changes which occur during aging since there are reports of increased synaptic cholesterohphospholipid ratios and decreased membrane fluidity in aged animals (1, 13, 15, 26, 31). Unless compensatory changes in membrane composition accompany the development of the age-related increase in synaptic cholesterol, one might expect to find decreased dopamine uptake in aged animals. In fact, synaptosomal uptake of
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436
dopamine is reportedly decreased in senescent versus mature mice (18). However, compensatory membrane changes may occur in rats since others have found no significant differences in dopamine uptake in aged rats (24,36).
AND DRUSE
Nonetheless, in light of the evidence of an age-related loss of striatal dopamine and dopaminergic receptors [reviewed in (19)], a cholesterol-induced decrease in dopamine uptake could potentially worsen dopaminergic function in the aged.
ACKNOWLEDGEMENTS
The authors would like to thank Dr. Allen Frankfater for the assistance he provided in the kinetic and fluorescence analyses and interpretation of the data.
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