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Phencyclidine: Effect on the accumulation of 3H-dopamine in synaptic vesicles
Life Sciences, Vol. 26, pp. 575-578 Printed in the U.S.A.
Pergamon
Press
PHENCYC~IDINE: EFFECT ON THE ACCUMULATION OF -H-DOPAMINE IN SYNAPTIC VESICLES T. E. Ary and H. L. Komiskey Washington State University College of Pharmacy Pullman, Washington 99164 (Received
in final form December
26, 1979)
Summary A subcellular fraction was prepared from pig caudate by ~ensity gradient centrifugation and characterized with respect to H-dopamine uptake. The fraction, containing synaptic storage ve~$cles, was shown to be dependent upon the presence of ATP and Mg in the incubation m~dium. Further, aliquots of the fraction isolated did accumulate H-DA with lin~arity up to an incubation time of ten minutes. Accumulation of H-DA was inhibited by reserpine (IC~o = 8.5 nM), a drug know~ to inhibit vesicular uptake of c a t e c h o l ~ i n e s . Accumulation of H-DA was reduced by the enantiomers of amphetamine. The S-(+)-enantiomer was i0 times more potent than the R-(-)-enantiomer. Phencyclidine w~s as potent as R-(-)-amphetamine in reducing the accumulation of H-dopamine. The effect of phencyclidine (PCP) on dopaminergic mechanisms are slowly being delineated. PCP has been reported to have no affinity for the dopamine ~eceptor (i).~ The drug does inhibit the synaptosomal accumulation of H-dopamine (~H-~A) (2, 3). However, part of the decrease in synaptosomal accumulation of -H-DA elicited bY310 ~M of PCP is probably caused by PCP releasing previously accumulated H-DA (4). Thus, PCP may affect the neuronal storage of dopamine. Since a large percentage of dopamine is thought to be stored in synaptic vesicles (5, 6, 7), the following experiments ~ere performed to determine the effects of PCP on the vesicular accumulation of -H-DA. Methods Isolation
of Synaptic
Vesicles
The synaptic vesicles were isolated by the technique of Segawa et al. (8) and Johnson et al. (9) as modified by Ferris and Tang (i0). The caudate nucleus was dissected from fresh pig brain and weighed. Then, the tissue was homogenized in i0 volumes of 0.32 M Sucrose - 25 mM Tris (pH 7.4 at 25°C) using i0 up and down strokes with the pestle revolving at 840 rpm. The teflon pestle (Kontes 886000) was modified so that the clearance between it and the vessel was 0.25 mm. The homogenate was centrifuged at 750 x g for i0 min at 4°C using a refrigerated centrifuge. The supernatant was then centrifuged at 15,000 x g for 15 min at 4°C using an ultracentrifuge. The pellet obtained was resuspended in 0.08 M sucrose (3 ml/g of original wet weight of caudate nucleus) by homogenizing for 90 seconds with the homogenizing pestle revolving at 840 rpm. The suspension was layered in polyallomer centrifuge tubes on a discontinuous sucrose density gradient. The order of layering of the sucrose density gradient 0024-3205/80/070575-04502.00/0 Copyright (c) 1980 Pergamon Press Ltd
576
Synaptic Vesicles
and Phencyclidine
Vol.
26, No.
7~
i i
was 12 ml of suspended pellet, 4.0 ml of 0.4 M sucrose, i0.0 ml of 0.6 M s ~ crose and ii.0 ml of 1.0 M sucrose. The gradient was then placed in a Bec:k~ SW27 rotor and centrifuged at 90,000 x g for 90 min at 4°C. The 0.4 M s ~ c ~ , layer was removed by aspiration with a syringe and needle and used as the source of synaptic vesicles. Incubations A 200 ul aliquot of the vesicle fraction was added to 480 ul of 0.~2 sucrose-25 mM Tris and i00 ul of 0.32 M sucrose-25 mM Tris containing 0.5 ~,iI ethylenediamine tetraacetic acid (EDTA). This solution was incubated for ~ : ~ at 25°C under 95%0^-5%C0^Z in an Eberbach metabolic water bath shaker. Foi;~ ing the 5 min incubation, MgSO 4 and ATP dissolved in 0.32 M sucrose - 25 m~,l Tris were added in 200 ul. All drugs were dissolved in 0.32 M sucrose - 25 ~ Tris and added to the incubation medium in lOul% After a 105sec preineubatJ~ : with or without drugs in the metabolic shaker, ~H-DA (6 x i~ dpm) was added the incubation medium in lOul. The final concentration of ~H-DA was 0.I ~.I i a final volume of 1.0 ml. The incubations were continued for 5 min at 25°C unless otherwise indicated. Each sample was then collected on ce!lu]ose acetate - cellulose nitrate membrane filters of 0.45 u pore size ( M i l l i p o r e ) vacuum filtration using a Millipore 1225 Sampling Manifold. The filters weJ~ washed twice with 3.5 ml of ice cold normal saline, transferred to counting vials, and dissolved in 1.0 ml of 2-methoxyethanol. Ten ml of scintillatio~ fluid (ACS, Amersham/Searle) was added and mixed thoroughly. Radioactivity the samples was measured in a liquid scintillation spectrometer. Each synaptic vesicle incubation sample was paired with a blank in<:,~i~ tion sample in order to determine the amount of non-specific binding of r ~ d i activity to the cellulose acetate-cellulose nitrate filter. Every blank s~:~ . was identical to its paired synaptic vesicle sample except that the synapti~ vesicle aliquot was replaced in the blank sample by an equal aliquot of b u ~ i The difference in radioactivity between a synaptic vesicle sample and its i ~ : ed blank sample was taken as a measure of the totaJ3amount of H-DA accum~J:~ tion in synaptic vesicl~s. Active accumulation of H-DA was determined by ~ ~ tracting the amount of H-DA accumulated by synaptic vesicles in the absen<:< : ATP and MgSO 4 from the amount of H-DA accumulated by synaptic vesicles in h:~ presence of ATP and MgSO 4.
method
Protein in the vesicular (ii).
preparations
was determined
by a dye-bindi~,y
Data Analysis The results obtained are reported as the mean values of n experime~l:~ with standard errorqof the means or as (ICON) values (that concentration cf drug which reduces JH-DA accumulation by 50%) with 95% confidence limits, i~ values were obtained by linear regression after transformation of the data ,, probits. Drugs Dopamine-2-3H (3H-DA) (25.3 or 30.0 Ci/mmol) was obtained from New En~ i : Nuclear, Boston, MA. The isomers of amphetamine were provided by Smith K]i:~ and French Laboratories, Phil., PA. Phencyclidine hydrochloride was obtai~,! from Applied Science Laboratories, State College, PA. Reserpine, dopamine hydrochloride, adenosine 5'-triphosphate (ATP), tris base and tris HCI were obtained from Sigma Chemical Co., St. Louis, MO.
Vol. 26, No. 7, 1980
Synaptic Vesicles and Phencyclidine
577
Results Initial experiments conducted at 25°C with ~arious concentrations of ATP and MgSO 4 showed that the maximum accumulation of JH-DA in 5 min occurred when 0.5 m M ATP and 1 m M MgSO, were used. Therefore, in all subsequent experiments 0.5 mM ATP and 1 mM MgSO. were used. The accumulation of H-DA at 25 C was found to be linear for i~ min. The accumulation of H-DA was les@ at 37°C and no accumulation occurred at 0-4°C. Hence, to ensure the maximum JH-DA accumulation with a linear relationship with time, the following vesicular incubations were performed for 5 min at 25°C. The influence of reserpine, 8-(+)- and R-(-)-amphetamine, and PCP on the accumulation of H-DA was determined. The IC~^ values for these^drugs are shown in Table i. Clearly, reserpine is the most p ~ e n t inhibitor of JH-DAaccumulation. S-(+)-amphetamine is about i0 times more potent than its R-(-)3~nantiomer, while PCP is equipotent to R-(-)-amphetamine as an inhibitor of H-DA accumulation. Table 1
Drug
IC50 (95% CL) ~M (0.0071-0.0099)
n
Reserpine
0.0085
S-(+)-amphetamine
4.5 (4.0-5.0)
7
R-(-)-amphetamine
37.0 (8.0-66.0)
6
Phencyclidine
33.0 (17.0-49.0)
6
6
IC50 values and 95% confidence limits for inhibitors of 3H-Dopamine accumulation in synaptic vesicles from pig caudate; 5 min incubation at 25°C; 0.5 mM ATP and 1.0 mM MgSO 4. Discussion The accumulation ~ 3H-DA into the subcellular fraction was examined for dependence on ATP and Mg , temperature, time S and inhibition by reserpine. In agreement with previous published studies on H-DA accumulation preparations containing synaptic vesicles, the accumulation of the 2~-amine was dependent on temperature, time and the concentration of ~TP and Mg . In addition,low doses of reserpine inhibited the accumulation of H-DA in the vesicular preparation. The conce n tr a t oi n s of ATP2$nd Mg 2+ used in this study are obviously lower than the 5 mM ATP and 33mM Mg used previously by other investigators measuring the accumulation of H-DA in pig caudate sy~$ptic vesicles (5). The incubation concentrations of 0.5 mM @TP and 1 mM Mg- were used in the present study because the accumulation of H-DA by the vesicular preparation was greatest at these concentrations. This discrepancy between the present and a previous study may be related to the procedure used to isolate the synaptic vesicles; Philippu and Beyer (5) purified synaptic vesicles using differential contrifugation techniques whereas in the present study the vesicles were purified using density gradient techniques. The data on the isomers of amphetamine provides additional support to the idea that the uptake of dopamine in noradrenergic and dopaminergic vesicles is similar (12). S-(+)-amphetamine has been reported to be I0 times more potent than its R-(-)-enantiomer as an inhibitor of -H-DA accumulation in vesicular preparations prepared from whole rat brain (i0). In the present study of
578
Synaptic Vesicles and Phencyclidine
Vol. 26, No. 7, 1980
3H-DA accumulation in synaptic vesicles purified from a predominantly dopaminergic region, the same ten-fold potency difference was found with isomers of amphetamine. The principal finding of the p~esent study is that PCP is equipotent to R-(-)-amphetamine as an inhibitor of H-DA accumulation in pig caudate synaptic vesicles. It should be pointed out that PCP has been reported to inhibit the accumulation of H-DA in rat striatal synaptosomes with a K. value (2) over 80 • i fold less than the IC50 value obtained in the present study. Presently, no one has determined whether PCP, at d~ses less than i0 ~M, decreases the synaptosomal or vesicular accumul~tion of H-DA by uptake inhibition and/or by releasing previously accumulated ~H-DA. However,3the apparent difference in the ability of PCP to decrease the accumulation of H-DA in synaptosomal and vesicular preparations, i~dicates that PCP is a more potent inhibitor of uptake and/or releaser of ~H-DA at the synaptic membrane level than in synaptic vesicles. Acknowledgements This investigation was supported in part by funds provided for medical and biological research by the State of Washington Initiative Measure No. 171. References i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12.
J.P. VINCENT, D. CAVEY, J.M. KAMENKA, P. GENESTE, and M. KAZDUNSKI, Brain Research 152, 176-182 (1978). R.E. GAREY and R.G. HEATH, Life Sciences 18, 1105-1110 (1976). R.C. SMITH, H.Y. MELTZER, R.C. ARORA, and J.M. DAVIS, Biochem. Pharmacol. 26, 1435-1439 (1977). T.E. ARY and H.LI KOMISKEY, The Pharmacologist 21, 241 (1979). A. PHILIPPU and J. BEYER, Naunyn-Schmiedeberg's Arch. Pharmacol. 278, 387-402 (1973). T.A. SLOTKIN, "Neuropoisons: Their Pat hophysio!ogica ! Actions" Vol. 2 (Ed. L.L° Simpson and D.R. Curtis) pp. 1-60, Plenum, New York (1974). L.L. IVERSON, Scientific American, 241, 134-150 (1979). T. SEGAWA, I. KURUMA, K. TAKATSUKA, and H. TAKAGI, J. Pharm. Pharmacol.20, 800-801 (1968). G.A. JOHNSON, S.J. BOUKMA, R.A. LAHTI, AND J. MATHEWS, J, Neurochem. 20, 1387-1392 (1973). R.M. FERRIS and F.L.M. TANG, J. Pharmacol. Exp. Ther. 210, 422-428 (1979). M.M. BRADFORD, Anal. Biochem. 72, 248-254 (1976). T.A. SLOTKIN, F.J. SEIDLER, W.L. WHITMORE, C. LAU, M. SALVAGGIO, and D.F. KIRKSEY, J. Neurochem. 31, 961-968 (1978).
Pergamon
Press
PHENCYC~IDINE: EFFECT ON THE ACCUMULATION OF -H-DOPAMINE IN SYNAPTIC VESICLES T. E. Ary and H. L. Komiskey Washington State University College of Pharmacy Pullman, Washington 99164 (Received
in final form December
26, 1979)
Summary A subcellular fraction was prepared from pig caudate by ~ensity gradient centrifugation and characterized with respect to H-dopamine uptake. The fraction, containing synaptic storage ve~$cles, was shown to be dependent upon the presence of ATP and Mg in the incubation m~dium. Further, aliquots of the fraction isolated did accumulate H-DA with lin~arity up to an incubation time of ten minutes. Accumulation of H-DA was inhibited by reserpine (IC~o = 8.5 nM), a drug know~ to inhibit vesicular uptake of c a t e c h o l ~ i n e s . Accumulation of H-DA was reduced by the enantiomers of amphetamine. The S-(+)-enantiomer was i0 times more potent than the R-(-)-enantiomer. Phencyclidine w~s as potent as R-(-)-amphetamine in reducing the accumulation of H-dopamine. The effect of phencyclidine (PCP) on dopaminergic mechanisms are slowly being delineated. PCP has been reported to have no affinity for the dopamine ~eceptor (i).~ The drug does inhibit the synaptosomal accumulation of H-dopamine (~H-~A) (2, 3). However, part of the decrease in synaptosomal accumulation of -H-DA elicited bY310 ~M of PCP is probably caused by PCP releasing previously accumulated H-DA (4). Thus, PCP may affect the neuronal storage of dopamine. Since a large percentage of dopamine is thought to be stored in synaptic vesicles (5, 6, 7), the following experiments ~ere performed to determine the effects of PCP on the vesicular accumulation of -H-DA. Methods Isolation
of Synaptic
Vesicles
The synaptic vesicles were isolated by the technique of Segawa et al. (8) and Johnson et al. (9) as modified by Ferris and Tang (i0). The caudate nucleus was dissected from fresh pig brain and weighed. Then, the tissue was homogenized in i0 volumes of 0.32 M Sucrose - 25 mM Tris (pH 7.4 at 25°C) using i0 up and down strokes with the pestle revolving at 840 rpm. The teflon pestle (Kontes 886000) was modified so that the clearance between it and the vessel was 0.25 mm. The homogenate was centrifuged at 750 x g for i0 min at 4°C using a refrigerated centrifuge. The supernatant was then centrifuged at 15,000 x g for 15 min at 4°C using an ultracentrifuge. The pellet obtained was resuspended in 0.08 M sucrose (3 ml/g of original wet weight of caudate nucleus) by homogenizing for 90 seconds with the homogenizing pestle revolving at 840 rpm. The suspension was layered in polyallomer centrifuge tubes on a discontinuous sucrose density gradient. The order of layering of the sucrose density gradient 0024-3205/80/070575-04502.00/0 Copyright (c) 1980 Pergamon Press Ltd
576
Synaptic Vesicles
and Phencyclidine
Vol.
26, No.
7~
i i
was 12 ml of suspended pellet, 4.0 ml of 0.4 M sucrose, i0.0 ml of 0.6 M s ~ crose and ii.0 ml of 1.0 M sucrose. The gradient was then placed in a Bec:k~ SW27 rotor and centrifuged at 90,000 x g for 90 min at 4°C. The 0.4 M s ~ c ~ , layer was removed by aspiration with a syringe and needle and used as the source of synaptic vesicles. Incubations A 200 ul aliquot of the vesicle fraction was added to 480 ul of 0.~2 sucrose-25 mM Tris and i00 ul of 0.32 M sucrose-25 mM Tris containing 0.5 ~,iI ethylenediamine tetraacetic acid (EDTA). This solution was incubated for ~ : ~ at 25°C under 95%0^-5%C0^Z in an Eberbach metabolic water bath shaker. Foi;~ ing the 5 min incubation, MgSO 4 and ATP dissolved in 0.32 M sucrose - 25 m~,l Tris were added in 200 ul. All drugs were dissolved in 0.32 M sucrose - 25 ~ Tris and added to the incubation medium in lOul% After a 105sec preineubatJ~ : with or without drugs in the metabolic shaker, ~H-DA (6 x i~ dpm) was added the incubation medium in lOul. The final concentration of ~H-DA was 0.I ~.I i a final volume of 1.0 ml. The incubations were continued for 5 min at 25°C unless otherwise indicated. Each sample was then collected on ce!lu]ose acetate - cellulose nitrate membrane filters of 0.45 u pore size ( M i l l i p o r e ) vacuum filtration using a Millipore 1225 Sampling Manifold. The filters weJ~ washed twice with 3.5 ml of ice cold normal saline, transferred to counting vials, and dissolved in 1.0 ml of 2-methoxyethanol. Ten ml of scintillatio~ fluid (ACS, Amersham/Searle) was added and mixed thoroughly. Radioactivity the samples was measured in a liquid scintillation spectrometer. Each synaptic vesicle incubation sample was paired with a blank in<:,~i~ tion sample in order to determine the amount of non-specific binding of r ~ d i activity to the cellulose acetate-cellulose nitrate filter. Every blank s~:~ . was identical to its paired synaptic vesicle sample except that the synapti~ vesicle aliquot was replaced in the blank sample by an equal aliquot of b u ~ i The difference in radioactivity between a synaptic vesicle sample and its i ~ : ed blank sample was taken as a measure of the totaJ3amount of H-DA accum~J:~ tion in synaptic vesicl~s. Active accumulation of H-DA was determined by ~ ~ tracting the amount of H-DA accumulated by synaptic vesicles in the absen<:< : ATP and MgSO 4 from the amount of H-DA accumulated by synaptic vesicles in h:~ presence of ATP and MgSO 4.
method
Protein in the vesicular (ii).
preparations
was determined
by a dye-bindi~,y
Data Analysis The results obtained are reported as the mean values of n experime~l:~ with standard errorqof the means or as (ICON) values (that concentration cf drug which reduces JH-DA accumulation by 50%) with 95% confidence limits, i~ values were obtained by linear regression after transformation of the data ,, probits. Drugs Dopamine-2-3H (3H-DA) (25.3 or 30.0 Ci/mmol) was obtained from New En~ i : Nuclear, Boston, MA. The isomers of amphetamine were provided by Smith K]i:~ and French Laboratories, Phil., PA. Phencyclidine hydrochloride was obtai~,! from Applied Science Laboratories, State College, PA. Reserpine, dopamine hydrochloride, adenosine 5'-triphosphate (ATP), tris base and tris HCI were obtained from Sigma Chemical Co., St. Louis, MO.
Vol. 26, No. 7, 1980
Synaptic Vesicles and Phencyclidine
577
Results Initial experiments conducted at 25°C with ~arious concentrations of ATP and MgSO 4 showed that the maximum accumulation of JH-DA in 5 min occurred when 0.5 m M ATP and 1 m M MgSO, were used. Therefore, in all subsequent experiments 0.5 mM ATP and 1 mM MgSO. were used. The accumulation of H-DA at 25 C was found to be linear for i~ min. The accumulation of H-DA was les@ at 37°C and no accumulation occurred at 0-4°C. Hence, to ensure the maximum JH-DA accumulation with a linear relationship with time, the following vesicular incubations were performed for 5 min at 25°C. The influence of reserpine, 8-(+)- and R-(-)-amphetamine, and PCP on the accumulation of H-DA was determined. The IC~^ values for these^drugs are shown in Table i. Clearly, reserpine is the most p ~ e n t inhibitor of JH-DAaccumulation. S-(+)-amphetamine is about i0 times more potent than its R-(-)3~nantiomer, while PCP is equipotent to R-(-)-amphetamine as an inhibitor of H-DA accumulation. Table 1
Drug
IC50 (95% CL) ~M (0.0071-0.0099)
n
Reserpine
0.0085
S-(+)-amphetamine
4.5 (4.0-5.0)
7
R-(-)-amphetamine
37.0 (8.0-66.0)
6
Phencyclidine
33.0 (17.0-49.0)
6
6
IC50 values and 95% confidence limits for inhibitors of 3H-Dopamine accumulation in synaptic vesicles from pig caudate; 5 min incubation at 25°C; 0.5 mM ATP and 1.0 mM MgSO 4. Discussion The accumulation ~ 3H-DA into the subcellular fraction was examined for dependence on ATP and Mg , temperature, time S and inhibition by reserpine. In agreement with previous published studies on H-DA accumulation preparations containing synaptic vesicles, the accumulation of the 2~-amine was dependent on temperature, time and the concentration of ~TP and Mg . In addition,low doses of reserpine inhibited the accumulation of H-DA in the vesicular preparation. The conce n tr a t oi n s of ATP2$nd Mg 2+ used in this study are obviously lower than the 5 mM ATP and 33mM Mg used previously by other investigators measuring the accumulation of H-DA in pig caudate sy~$ptic vesicles (5). The incubation concentrations of 0.5 mM @TP and 1 mM Mg- were used in the present study because the accumulation of H-DA by the vesicular preparation was greatest at these concentrations. This discrepancy between the present and a previous study may be related to the procedure used to isolate the synaptic vesicles; Philippu and Beyer (5) purified synaptic vesicles using differential contrifugation techniques whereas in the present study the vesicles were purified using density gradient techniques. The data on the isomers of amphetamine provides additional support to the idea that the uptake of dopamine in noradrenergic and dopaminergic vesicles is similar (12). S-(+)-amphetamine has been reported to be I0 times more potent than its R-(-)-enantiomer as an inhibitor of -H-DA accumulation in vesicular preparations prepared from whole rat brain (i0). In the present study of
578
Synaptic Vesicles and Phencyclidine
Vol. 26, No. 7, 1980
3H-DA accumulation in synaptic vesicles purified from a predominantly dopaminergic region, the same ten-fold potency difference was found with isomers of amphetamine. The principal finding of the p~esent study is that PCP is equipotent to R-(-)-amphetamine as an inhibitor of H-DA accumulation in pig caudate synaptic vesicles. It should be pointed out that PCP has been reported to inhibit the accumulation of H-DA in rat striatal synaptosomes with a K. value (2) over 80 • i fold less than the IC50 value obtained in the present study. Presently, no one has determined whether PCP, at d~ses less than i0 ~M, decreases the synaptosomal or vesicular accumul~tion of H-DA by uptake inhibition and/or by releasing previously accumulated ~H-DA. However,3the apparent difference in the ability of PCP to decrease the accumulation of H-DA in synaptosomal and vesicular preparations, i~dicates that PCP is a more potent inhibitor of uptake and/or releaser of ~H-DA at the synaptic membrane level than in synaptic vesicles. Acknowledgements This investigation was supported in part by funds provided for medical and biological research by the State of Washington Initiative Measure No. 171. References i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12.
J.P. VINCENT, D. CAVEY, J.M. KAMENKA, P. GENESTE, and M. KAZDUNSKI, Brain Research 152, 176-182 (1978). R.E. GAREY and R.G. HEATH, Life Sciences 18, 1105-1110 (1976). R.C. SMITH, H.Y. MELTZER, R.C. ARORA, and J.M. DAVIS, Biochem. Pharmacol. 26, 1435-1439 (1977). T.E. ARY and H.LI KOMISKEY, The Pharmacologist 21, 241 (1979). A. PHILIPPU and J. BEYER, Naunyn-Schmiedeberg's Arch. Pharmacol. 278, 387-402 (1973). T.A. SLOTKIN, "Neuropoisons: Their Pat hophysio!ogica ! Actions" Vol. 2 (Ed. L.L° Simpson and D.R. Curtis) pp. 1-60, Plenum, New York (1974). L.L. IVERSON, Scientific American, 241, 134-150 (1979). T. SEGAWA, I. KURUMA, K. TAKATSUKA, and H. TAKAGI, J. Pharm. Pharmacol.20, 800-801 (1968). G.A. JOHNSON, S.J. BOUKMA, R.A. LAHTI, AND J. MATHEWS, J, Neurochem. 20, 1387-1392 (1973). R.M. FERRIS and F.L.M. TANG, J. Pharmacol. Exp. Ther. 210, 422-428 (1979). M.M. BRADFORD, Anal. Biochem. 72, 248-254 (1976). T.A. SLOTKIN, F.J. SEIDLER, W.L. WHITMORE, C. LAU, M. SALVAGGIO, and D.F. KIRKSEY, J. Neurochem. 31, 961-968 (1978).