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Utilization of 3H-dopamine as a photoaffinity label of brain synaptosomes
Pergamon Press
Life Sciences, Vol. 26, pp . 85-88 Printed in the U.S .A .
UTILIZATION ,OF 3H-DOPAMINE AS A PHOTOAFFINITY LABEL OF BRAIN SYNAPTOSOMES Bruce Davies, Leo Abood and *Andrew M. Tometsko Department of Biochemistry and Center for Brain Research University of Rochester Medical Center Rochester, New York 14642 and *Litron Laboratories Limited Rochester, New York (Received in final form November 16, 1979) Stmmmary The possibility of utilizing 3 H-dopamine ae a photoaffinity label for rat brain synaptosomes is investigated . 3H-dopamine interacts covalently with intact and disrupted synaptosames in the absence of light, but the interaction increases as much as fourfold following flash photolytis with ultraviolet light. The photolytic interaction is intact, but not disrupted, synaptosomes is inhibited by benzotropine and cocaine, but not by haloperidol . It is concluded that a major photolytic reactive site in synaptosomes ie that associated with dopamine reuptake . The highly reactive nature of catecholamine oxidation products has led to their u~e in affinity labeling experiments (1-3) . Maguire et _al . (1) used the products of 3ü~norepinephrine oxidation to label canine particulate fractions, and found that in the absence of a reducing agent, labeling levels were very high . It was also suggested that this binding might be a significant undesirable contribution to specific binding experiments . Catachol-O~methyltranaferase (COMT) has been selectively inactivated by covalent binding of 6hydroxydopamine oxidation products (2) . The inactivation could be prevented by including substrates of COMT during the labeling reactions . Saner _et _al . (3) demonstrated the covalent attachment of dopamine (DA) oxidation products to bovine serum albumin (BSA) . The attachment was dependent both on time and 3 H-DA concentration. Acetylation of the nucleophilic groups greatly reduced binding, indicating a direct chemical involvement of the groups . Takayanagi _et al . (4) reported the use of photolytic generated oxidation products of isoprotereaol as photoaffinity labels of ß-adrenergic receptors on guinea pig taeaia . Addition of the ß-adrenergic receptor antagonist propranolol protected the system from photolytic inactivation by isoproterenol . Since DA is readily oxidized both chemically and photolytically, experiment~ were undertaken to sanest its possible use as e photoaffinity label . The system used to test this possibility is the DA-reuptake mechanism in rat brain syaaptosomes . DA ie rapidly transported (5-7) by this system as a method of terminating neural tranamitsioa . Rat brains, excluding cerebella, were washed is 0 .32 M sucrose and homogenized is 10 volumes of 0.32 M sucrose is a modified (0 .25 mm clearance) The homogenate was centrifuged at teflon-glass homogenizer using 4 strokes . 0024-3205/80/020085-04$02 .00/0 Copyright (c) 1980 Pergamon Preee Ltd
86
Dopamine as a Photoaffinity Label
Vol . 26, No . 2, 1980
1000 x g at 4°C and the resulting supernatant centrifuged at 20,000 x g to yield a crude mitochondrial fraction, which was resuspended in 0 .32 C sucrose (CMF) . A portion of this fraction was frozen at -20 ° C and used to prepare membranes . After repeated freeze-thawing (3x), the preparation was centrifuged at 25,000 x g and washed (3x) with 5 mM Tris HCl pH 7 .4 containing 1 mM ascorbic acid . Photolysis experiments were carried out using a model 457 Micropulser (Xenon Corp .), which powers a refrigerated (4 ° C) and air cooled N-725C linear quartz flashtube rated at 25 joules per pulse . Photolysis was carried out for 10 min (14 pulses per second) at 4° C. The samples (2-4 ml) were photolysed while being stirred in 1 x 2.5 covered polystyrene petri dishes at a distance of 2 cm . The synaptosome containing CMF (150 mg/ml wet wt) was added to a modified Krebs-Ringer phosphate buffer pH 7 .4 containing 92 mM NaCl, 3.7 mM KC1, 0 .53mM CaClp, 1 mM KH2POy , 1 mM MgSO y , 19 .5 mM Na 2HP0y , 1 mM glucose, 1 mM ascorbic acid and 12 .5 uM pargyline . This buffer also contained the inhibitor at the desired concentration. After the synaptosome solution was preincubated for 5 min on ice, 3H-DA (10.02 Ci/mM in 1 mM ascorbic acid) was added to a final conc . of 1 x 10-~M and incubation continued for 5 min on ice . The sample was then photolysed as described or kept on ice in the dark . Immediately following photolysis, an equal volume of 1% BSA in 0 .32 M sucrose was added and the reaction mixture incubated for 20 min on ice. Synaptosomes were centrifuged in a Brinkman model 3200 microfuge and the supernatant discarded. After the addition of an equal volume of double distilled water to lyse the synaptosomes, the membranes were isolated and washed with resuspension of the intermediate pellet (3x) with double distilled water. Proteins were precipitated from the washed membranes by addition of 1N trichloroacetic acid (TCA) and pelleted in the microfuge . The washing procedure in 1N TCA was repeated twice . The final pellet was solubilized in 1N NaOH and the radioactivity was determined by liquid scintillation counting . Membranes (125 mg/ml wet wt) were labeled in essentially the same manner except for a few differences . These included using 5 mM Tris-1 mM ascorbic acid, 3H-DA (45 Ci/mM, final conc . 24 nM), 3% TCA to precipitate the proteins and 1% sodium dodecyl sulfate to solubilize the membranes. Photolysis of 3H-DA to BSA was conducted in a similar method, incubating a solution of BSA in 5 mM Tris HC1 + 1 mM ascorbic acid for 5 min with 3H-DA (10.05 Ci/mM) at a final concentration of 1 x 10 -~M, on ice prior to photolysis . The labeled BSA was precipitated by addition of 1N TCA, dissolved in 0 .5 M NaOH, and then reprecipitated in 1N TCA, a procedure repeated twice with final solubilizatioa in 0.5 M NaOH . The amount of 3H-DA bound was determined by liquid scintillation counting . TABLE I 3 H-Dopamine Reaction with Bovine Serum Albumin Condition
3H-DA Bound pmolea/mg
Light
3 .445 ± 0.18
Dark
0.816 ± 0 .03
(The results are an average of 3 experiments) The data in Table 2 indicate that photolysis greatly increased (ti4x) the amount of 3A-DA reacting with BSA (this indicates that covalently reactive oxidation products can be generated by photolyses in amounts higher than those forming through simple chemical oxidation) . The synaptosomes (Table II) also showed significant increases in the labeling of photolysed samples over dark controls (P < 0.001) . In the presence of either benztropine or cocaine, which
Vol . 26, No . 2, 1980
Dopamine as a Photoaffinity Label
87
are inhibitors of DA uptake, there occurred a significant decrease in the labeling with 3H-DA . TABLE II Effect of Various Paychotropic Drugs on 3H-Dopamine Reaction with Synaptosomes after Photolysis Synaptosomes
Conditions 3H-DA Bound fmoles/mg wet weight
X og Light Control
Membranes Range to Light Control
3H-DA Bound CPM
Light
4 .21 ± 0 .05
100
Light ~ Benz 1 x 10 -6M
1.61 ± 0 .04
38
(38-90) 73 .2 ± 21
5121 ± 284
Light ~ Cocaine 1.94 ± 0 .06 1 x 10 -6 M
46
(46-73) 59 .5 ± 19
5491 ± 371
Light 6 Halo 1 x 10- ~M
4 .41 ± 0.07
104
(97-126) 114 ± 12
4966 ± 545
Dark
0 .98 ± 0.05
23
(23-82) 63 ± 22
4230 ± 110
5043 t 392
The data represent a single typical experiment run in triplicate and expressed as mean t standard deviation . CPM ~ counts/min radioactivity . The data in column 3 represent the range and mean values as X of light control and are based on 5-10 separate experiments . Haloperidol, a poet-synaptic receptor blocker, was without significant effect . The protection afforded by benztropine or cocaine indicates that the membrane component most susceptible to covalent DA interaction is associated with the reuptake site . In order to exclude the possibility that the benztropine or cocaine interfered with photolytic oxidation _~t the site, a control experiment was performed interposing a solution of benztropine of the same After concentration as a filter in front of the synaptosome reaction mixture . photolysis, the levels of 3H-DA bound to this preparation were not signifiTo eliminate the possibility that cantly different from photolysed control. a photo-generated species of the tropine residue is responsible for the inhibition of DA binding, atropine, a tropine molecule with no effect on reuptake, No inhibitory effect was noted. While benzwas tested against DA binding. tropine and cocaine reduce the interaction of 3H-DA with synaptosomes they have no significant effect on membranes . It would appear,~therefore, that the covalent interaction of photolysed DA requires an intact functional synaptosome . Since the difference between photolysed and dark labeling is greater in synaptosomes than in membrane fragments, it would appear that the outer surface of the synaptosome is less susceptible to dark reactions than the inner surface . Imipramine, a potent inhibitor of norepinephrine reuptake, was also without effect on DA interaction . It should be mentioned that, from experiment to experiment, some variation occurred in the magnitude of the covalent interaction of DA with synaptosomes
88
Dopamine as a Photoaffiaity Label
Vol . 26, No . 2, 1980
both photolysed and dark . The greatest variability seems to be in the dark control and may be attributable to the conditions of homogenization and experimental time requirements . The beat and moat consistent results were obtained A likely with a teflon-glass homogenizer modified to a clearance 0.25 mm. explanation is that the integrity and viability of the synaptosomea influences the level of labeling . It can be shown, for example, that even when stored in ice cold isotonic sucrose, DA uptake of synaptosomea rapidly decreases with time . As indicated in a total of 10 separate experiments performed, comparable light-dark differences to those reported in Table II were observed with intact synaptosomea . Acknowledgements This research was supported by a USPH grant DA 00464 and a grant from Philip Morris . The senior author was supported by NIH Training Grant GMO 7230-05 . We would like to thank McNeil Laboratories for their generous donation of haloperidol and Merck, Sharp and Dohme for their generous donation of benztropine meaylate . References 1. 2. 3. 4. 5. 6. 7.
M.E . MAGUIRE, P .H . GOLDMAN, and A. G . GILMAN, Molecular Pharmacology 10 563-581 (1974) . R.T. BORCHARDT, E .E . SMISSMAN, D . NERLAND, and J.R . REID, _J . _Mad . Chem . _19 30-36 (1976) . A . SANER, and H. THOENEN, Molecular Pharmacology _7 147-154 (1970) . I . TAKAYANAGI, M. YOSHIORA, K. TARAGI, and Z . TAMURA, _Eur . _J . Pharm . _35 121-125 (1976) . S .H . SNYDER, J .T . COYLE, J. Pharm. Ex~. Ther . _165 78-85 (1969) . A . RANDRUP, C. BRAESTRUP, Psychopharmacology _53 309-314 (1977) . J .E . HARRIS, R.J . BALDESSARINI, Life Sciences 13 303-312 (1973) .
Life Sciences, Vol. 26, pp . 85-88 Printed in the U.S .A .
UTILIZATION ,OF 3H-DOPAMINE AS A PHOTOAFFINITY LABEL OF BRAIN SYNAPTOSOMES Bruce Davies, Leo Abood and *Andrew M. Tometsko Department of Biochemistry and Center for Brain Research University of Rochester Medical Center Rochester, New York 14642 and *Litron Laboratories Limited Rochester, New York (Received in final form November 16, 1979) Stmmmary The possibility of utilizing 3 H-dopamine ae a photoaffinity label for rat brain synaptosomes is investigated . 3H-dopamine interacts covalently with intact and disrupted synaptosames in the absence of light, but the interaction increases as much as fourfold following flash photolytis with ultraviolet light. The photolytic interaction is intact, but not disrupted, synaptosomes is inhibited by benzotropine and cocaine, but not by haloperidol . It is concluded that a major photolytic reactive site in synaptosomes ie that associated with dopamine reuptake . The highly reactive nature of catecholamine oxidation products has led to their u~e in affinity labeling experiments (1-3) . Maguire et _al . (1) used the products of 3ü~norepinephrine oxidation to label canine particulate fractions, and found that in the absence of a reducing agent, labeling levels were very high . It was also suggested that this binding might be a significant undesirable contribution to specific binding experiments . Catachol-O~methyltranaferase (COMT) has been selectively inactivated by covalent binding of 6hydroxydopamine oxidation products (2) . The inactivation could be prevented by including substrates of COMT during the labeling reactions . Saner _et _al . (3) demonstrated the covalent attachment of dopamine (DA) oxidation products to bovine serum albumin (BSA) . The attachment was dependent both on time and 3 H-DA concentration. Acetylation of the nucleophilic groups greatly reduced binding, indicating a direct chemical involvement of the groups . Takayanagi _et al . (4) reported the use of photolytic generated oxidation products of isoprotereaol as photoaffinity labels of ß-adrenergic receptors on guinea pig taeaia . Addition of the ß-adrenergic receptor antagonist propranolol protected the system from photolytic inactivation by isoproterenol . Since DA is readily oxidized both chemically and photolytically, experiment~ were undertaken to sanest its possible use as e photoaffinity label . The system used to test this possibility is the DA-reuptake mechanism in rat brain syaaptosomes . DA ie rapidly transported (5-7) by this system as a method of terminating neural tranamitsioa . Rat brains, excluding cerebella, were washed is 0 .32 M sucrose and homogenized is 10 volumes of 0.32 M sucrose is a modified (0 .25 mm clearance) The homogenate was centrifuged at teflon-glass homogenizer using 4 strokes . 0024-3205/80/020085-04$02 .00/0 Copyright (c) 1980 Pergamon Preee Ltd
86
Dopamine as a Photoaffinity Label
Vol . 26, No . 2, 1980
1000 x g at 4°C and the resulting supernatant centrifuged at 20,000 x g to yield a crude mitochondrial fraction, which was resuspended in 0 .32 C sucrose (CMF) . A portion of this fraction was frozen at -20 ° C and used to prepare membranes . After repeated freeze-thawing (3x), the preparation was centrifuged at 25,000 x g and washed (3x) with 5 mM Tris HCl pH 7 .4 containing 1 mM ascorbic acid . Photolysis experiments were carried out using a model 457 Micropulser (Xenon Corp .), which powers a refrigerated (4 ° C) and air cooled N-725C linear quartz flashtube rated at 25 joules per pulse . Photolysis was carried out for 10 min (14 pulses per second) at 4° C. The samples (2-4 ml) were photolysed while being stirred in 1 x 2.5 covered polystyrene petri dishes at a distance of 2 cm . The synaptosome containing CMF (150 mg/ml wet wt) was added to a modified Krebs-Ringer phosphate buffer pH 7 .4 containing 92 mM NaCl, 3.7 mM KC1, 0 .53mM CaClp, 1 mM KH2POy , 1 mM MgSO y , 19 .5 mM Na 2HP0y , 1 mM glucose, 1 mM ascorbic acid and 12 .5 uM pargyline . This buffer also contained the inhibitor at the desired concentration. After the synaptosome solution was preincubated for 5 min on ice, 3H-DA (10.02 Ci/mM in 1 mM ascorbic acid) was added to a final conc . of 1 x 10-~M and incubation continued for 5 min on ice . The sample was then photolysed as described or kept on ice in the dark . Immediately following photolysis, an equal volume of 1% BSA in 0 .32 M sucrose was added and the reaction mixture incubated for 20 min on ice. Synaptosomes were centrifuged in a Brinkman model 3200 microfuge and the supernatant discarded. After the addition of an equal volume of double distilled water to lyse the synaptosomes, the membranes were isolated and washed with resuspension of the intermediate pellet (3x) with double distilled water. Proteins were precipitated from the washed membranes by addition of 1N trichloroacetic acid (TCA) and pelleted in the microfuge . The washing procedure in 1N TCA was repeated twice . The final pellet was solubilized in 1N NaOH and the radioactivity was determined by liquid scintillation counting . Membranes (125 mg/ml wet wt) were labeled in essentially the same manner except for a few differences . These included using 5 mM Tris-1 mM ascorbic acid, 3H-DA (45 Ci/mM, final conc . 24 nM), 3% TCA to precipitate the proteins and 1% sodium dodecyl sulfate to solubilize the membranes. Photolysis of 3H-DA to BSA was conducted in a similar method, incubating a solution of BSA in 5 mM Tris HC1 + 1 mM ascorbic acid for 5 min with 3H-DA (10.05 Ci/mM) at a final concentration of 1 x 10 -~M, on ice prior to photolysis . The labeled BSA was precipitated by addition of 1N TCA, dissolved in 0 .5 M NaOH, and then reprecipitated in 1N TCA, a procedure repeated twice with final solubilizatioa in 0.5 M NaOH . The amount of 3H-DA bound was determined by liquid scintillation counting . TABLE I 3 H-Dopamine Reaction with Bovine Serum Albumin Condition
3H-DA Bound pmolea/mg
Light
3 .445 ± 0.18
Dark
0.816 ± 0 .03
(The results are an average of 3 experiments) The data in Table 2 indicate that photolysis greatly increased (ti4x) the amount of 3A-DA reacting with BSA (this indicates that covalently reactive oxidation products can be generated by photolyses in amounts higher than those forming through simple chemical oxidation) . The synaptosomes (Table II) also showed significant increases in the labeling of photolysed samples over dark controls (P < 0.001) . In the presence of either benztropine or cocaine, which
Vol . 26, No . 2, 1980
Dopamine as a Photoaffinity Label
87
are inhibitors of DA uptake, there occurred a significant decrease in the labeling with 3H-DA . TABLE II Effect of Various Paychotropic Drugs on 3H-Dopamine Reaction with Synaptosomes after Photolysis Synaptosomes
Conditions 3H-DA Bound fmoles/mg wet weight
X og Light Control
Membranes Range to Light Control
3H-DA Bound CPM
Light
4 .21 ± 0 .05
100
Light ~ Benz 1 x 10 -6M
1.61 ± 0 .04
38
(38-90) 73 .2 ± 21
5121 ± 284
Light ~ Cocaine 1.94 ± 0 .06 1 x 10 -6 M
46
(46-73) 59 .5 ± 19
5491 ± 371
Light 6 Halo 1 x 10- ~M
4 .41 ± 0.07
104
(97-126) 114 ± 12
4966 ± 545
Dark
0 .98 ± 0.05
23
(23-82) 63 ± 22
4230 ± 110
5043 t 392
The data represent a single typical experiment run in triplicate and expressed as mean t standard deviation . CPM ~ counts/min radioactivity . The data in column 3 represent the range and mean values as X of light control and are based on 5-10 separate experiments . Haloperidol, a poet-synaptic receptor blocker, was without significant effect . The protection afforded by benztropine or cocaine indicates that the membrane component most susceptible to covalent DA interaction is associated with the reuptake site . In order to exclude the possibility that the benztropine or cocaine interfered with photolytic oxidation _~t the site, a control experiment was performed interposing a solution of benztropine of the same After concentration as a filter in front of the synaptosome reaction mixture . photolysis, the levels of 3H-DA bound to this preparation were not signifiTo eliminate the possibility that cantly different from photolysed control. a photo-generated species of the tropine residue is responsible for the inhibition of DA binding, atropine, a tropine molecule with no effect on reuptake, No inhibitory effect was noted. While benzwas tested against DA binding. tropine and cocaine reduce the interaction of 3H-DA with synaptosomes they have no significant effect on membranes . It would appear,~therefore, that the covalent interaction of photolysed DA requires an intact functional synaptosome . Since the difference between photolysed and dark labeling is greater in synaptosomes than in membrane fragments, it would appear that the outer surface of the synaptosome is less susceptible to dark reactions than the inner surface . Imipramine, a potent inhibitor of norepinephrine reuptake, was also without effect on DA interaction . It should be mentioned that, from experiment to experiment, some variation occurred in the magnitude of the covalent interaction of DA with synaptosomes
88
Dopamine as a Photoaffiaity Label
Vol . 26, No . 2, 1980
both photolysed and dark . The greatest variability seems to be in the dark control and may be attributable to the conditions of homogenization and experimental time requirements . The beat and moat consistent results were obtained A likely with a teflon-glass homogenizer modified to a clearance 0.25 mm. explanation is that the integrity and viability of the synaptosomea influences the level of labeling . It can be shown, for example, that even when stored in ice cold isotonic sucrose, DA uptake of synaptosomea rapidly decreases with time . As indicated in a total of 10 separate experiments performed, comparable light-dark differences to those reported in Table II were observed with intact synaptosomea . Acknowledgements This research was supported by a USPH grant DA 00464 and a grant from Philip Morris . The senior author was supported by NIH Training Grant GMO 7230-05 . We would like to thank McNeil Laboratories for their generous donation of haloperidol and Merck, Sharp and Dohme for their generous donation of benztropine meaylate . References 1. 2. 3. 4. 5. 6. 7.
M.E . MAGUIRE, P .H . GOLDMAN, and A. G . GILMAN, Molecular Pharmacology 10 563-581 (1974) . R.T. BORCHARDT, E .E . SMISSMAN, D . NERLAND, and J.R . REID, _J . _Mad . Chem . _19 30-36 (1976) . A . SANER, and H. THOENEN, Molecular Pharmacology _7 147-154 (1970) . I . TAKAYANAGI, M. YOSHIORA, K. TARAGI, and Z . TAMURA, _Eur . _J . Pharm . _35 121-125 (1976) . S .H . SNYDER, J .T . COYLE, J. Pharm. Ex~. Ther . _165 78-85 (1969) . A . RANDRUP, C. BRAESTRUP, Psychopharmacology _53 309-314 (1977) . J .E . HARRIS, R.J . BALDESSARINI, Life Sciences 13 303-312 (1973) .