invasive presurgical recordings. Careful evaluation is still needed to establish MEG as a tool for routine clinical use. This study was financially supported by the Academy of Finland and by the Sigrid Juselius Foundation. We thank the collaborative presurgical team at the Vaajasalo Hospital, Heleena Hurskainen for neuropsychological expertise, and Dr Leo Paljiirvi for comments on histology.
References 1. Barth DS, Sutherling WW, Engel JJ, Beatty J. Neuromagnetic localization of epileptiform spike activity in the human brain. Science 1982;2 18:891-894 2. Modena I, Ricci GB, Barbanera S, et al. Biomagnetic measurements of spontaneous brain activity in epileptic patients. Electroencephalogr Clin Neurophysiol 1982;54:622-628 3. Rose DF, Smith PD, Sat0 S. Magnetoencephalography and epilepsy research. Science 1987;238:329-3 3 5 4. Sutherling WW, Barth DS. Neocortical propagation in temporal lobe spike foci on magnetoencephalography and electroencephalography. Ann Neurol 1989;25 :3 73-38 1 5. Ricci GB, Romani GL, Salustri C, et al. Study of focal epilepsy by multichannel neuromagnetic measurements. Electroencephalogr Clin Neurophysiol 1987;66:358-368 6. Sutherling WW, Crandall PH, Engel JJ, et al. The magnetic field of complex partial seizures agrees with intracranial localizations. Ann Neurol 1987;21:548-558 7. Stefan H, Schneider S, Abraham-Fuchs K, et al. Application of a multichannel MEG system in temporal lobe epilepsy. In: Williamson SJ, Hoke M, Stroink G, Kotani M, eds. Advances in biomagnetism. New York Plenum, 1989:279-282 8. Tiihonen J, Hari R, Kajola M, et al. Localization of epileptic foci using a large-area magnetometer and functional brain anatomy. Ann Neurol 1990;27:283-290 9. Ahonen A, HimAilEnen M, Kajola M, et al. Multichannel SQUID systems for brain research. IEEE Trans Magn 1991; 27:2786-2792 10. Knuutila J, Ahlfors S, Ahonen A, et al. A large-area low-noise seven-channel DC SQUID magnetometer for brain research. Rev Sci Instr 1987;58:2145-2156 11. Hari R. The neuromagnetic method in the study of the human auditory cortex. In: Grandori F, Hoke M, Romani GL, eds. Auditory evoked magnetic fields and potentials. Advances in audiology. Base1 Karger, 1990:222-282 12 Williamson SJ, Kaufman L. Biomagnetism. J Magn Magn Mater 1981;22:129-202 13. Hari R, Lounasmaa OV. Recording and interpretation of cerebral magnetic fields. Science 1989;244:432-436
Parkmsonism-inducing Neurotoxin MPP': Uptake and Toxicity in Nonneuronal COS -Cells Expressing Dopamine Transporter cDNA S. Kitayama, DDS, PhD, S. Shimada, MD, and G. R. Uhl, MD, PhD
Expression of a cloned dopamine transporter complementary DNA in COS cells allows these primate kidney cells to accumulate the parkinsonism-inducing neurotoxin metabolite MPP+ (1-methyl-4-phenylpyridinium) avidly, and MPP+ toxicity results. By documenting that the dopamine transporter can confer MPP+ sensitivity to nonneural cells, these results highlight the key role that this transporter could play in mechanisms underlying parkinsonism. Kitayama S, Shimada S, Uhl GR. Parkinsonisminducing neurotoxin MPP+: uptake and toxicity in nonneuronal COS cells expressing dopamine transporter cDNA. Ann Neurol 1992;32:109-111
The dopamine transporter normally acts to terminate dopaminergic neurotransmission by sodium-dependent reaccumulation of dopamine into presynaptic neurons [l, 21.The transporter can also accumulate neurotoxins with structural features resembling dopamine; its ability to concentrate the parkinsonism-inducing toxin 1-methyl-4-phenylpyridinium (MPP' ) is required for N-methyl-4-phenyl-l,2,5,6-tetrahydropyridine (MPTP) neurotoxicity in vivo and in vitro, although other steps may also be involved [3-71. We recently cloned a functional complementary DNA (cDNA) encoding a dopamine transporter from a rat midbrain cDNA library using D N A homology and expression approaches [sf.DATl encodes a protein that can transport [3H}dopamine (E3H1DA) into Xenopus oocytes and COS cells and bind the cocaine analog { 3H]2p-carbmethoxy-3~-(4-fluorophenyl)-trope (r3H}CFT)with a pharmacological profile similar to From the Laboratory of Molecular Neurobiology, Addiction Research Center/National Institute on Drug Abuse, and the Departments of Neurology and Neuroscience, Johns Hopkins IJniversity School of Medicine, Baltimore, MD.
Received Nov 8, 1991, and in revised form Jan 7, 1992. Accepted for publication Jan 7, 1992. Address correspondence to Dr Uhl, Laboratory of Molecular Neurobiology, Addiction Research CenterlNational Institute on Drug Abuse, PO Box 5180, Baltimore, MD 21224.
Brief Communication: Kitayama et
al:
DA Transporter cDNA and MPP'
103
that of the native rat striatal transporter (81. Availability of this cDNA subcloned into a eukaryotic cell expression vector, pcDNAI (pcDNADAT I), allows assessment of the ability of this transporter to accumulate MPP+ into nonneural cells and of its ability to make accumulated MPP+ toxic to such cells.
70 _.
.
3H-DA pDATI control 3H-DA pDATl + IOpM (-) Cocaine
A 3H-DA pCDM8
0 3H-MPP+pDAT1 wntrol 3H-MPP+pDAT1 + lOpM (-1 C o ~ l l m , A 3H-MPP+pCDM8
Materials and Methods COS cells were transfected by electroporation with 20 pg/ lo7cells of pcDNADAT1, pCDM8, or pCDMbeta, as previously described IS, 91. Transfected COS cells were plated in 35-mm dishes in 2 mL Dulbecco's modified minimal essential medium containing 10% fetal bovine serum. Cells were either (1) cultured for 2 to 3 days in control medium, (2) grown in normal medium for 24 hours and changed to medium containing test drugs for 15 to 20 hours before harvesting, or (3) changed with normal medium 24 hours after transfection, to provide a control for (2). To assess neurotransmitter and neurotoxin uptake, cells were washed three times in Krebs-Ringer-HEPES (KRH) buffer at room temperature and incubated with 5 nmol/L C3H]MPPC (83.9 Ci/mmol, NEN [Dupont, Boston, MA)) or 10 nmol/L E3H]DA (50.0 Ci/mmol, Amersham, UK) in KRH buffer at 37°C. Uptake was terminated by three washes with ice-cold KRH, and radioactivity was extracted with 2 N NaOH and measured by scintillation counting. To test involvement of extracellular Na' in transport, NaCI in KRH was replaced by LiCl in some experiments. To assess MPP+ toxicity, cells and medium were assayed for: (1) lactic dehydrogenase (LDH) activity using a colorimetric procedure {lo]. Values presented represent the percent by which normalized LDH values released into the medium increased as a fraction of total (plate plus medium) LDH values. (2) r3H]CGP12177 (59 Ci/mmol, NEN) binding to beta-adrenergic receptors as previously described [ 1I]; and (3) C3H]CFT binding to dopamine transporters [l2, 13). Binding of 1 nmol/L 13H)CFT (82.7 Cilmmol, NEN) to intact cell membranes was assessed by incubation at 4°C for 2 hours in 1 mL KRH, three washes with 1 mL ice-cold buffer, extraction of radioactivity with NaOH, and scintillation counting. Nonspecific binding was defined as binding in the presence of 30 kmol/L { - ) cocaine. Statistical analyses were performed using Student's t-test.
Results and Discussion COS cells transfected with pcDNADATl displayed avid uptake of r3H}DA and E3H}MPP+ (Fig 1). Dopamine and MPP+ uptakes were rapid, sodiumand temperature-dependent, and cocaine-blockable [1-3,8} although E3H]DA uptake reached peak values more rapidly than I3H)MPP+ accumulation. COS cells do not accumulate the neurotoxin when transfected with control plasmids (see Fig 1). Expression of DATl can thus specifically confer MPP+ uptake on nonneural cells. Uptake displays interesting features. The failure of MPP+ uptake to saturate when DA transport has reached plateau values could point to differences in the intracellular sequestration or binding of the accumu110 Annals of Neurology
Vol 32 No I July 1992
0
5
10
15
20
Time (mln)
Fig 1. Time-course of f3H)MPP+ and t3H)DA uptake by COS cells transfected with pcDNADATl or vector only. Values are the means of triplicate determinations.
lated MPP', or to differences in its affinity for one of the states or forms of the transporter. Although such differences have not been clearly reported in synaptosomal studies [but see 141, these different properties of the two substrates, DA and MPP', leave open the possibility that pharmacological strategies for altering transporter function to selectively inhibit neurotoxin uptake but spare DA transport might be found. Expression of DATl conferred MPP' toxicity on transfected COS cells. Fifteen to 20 hours' exposure to lp,mol/L MPP+ increased LDH release and decreased (3H7CFT binding in pcDNADATl-expressing cultures (Fig 2A,B). Changes in each of these two markers were almost absent when 0.1 p,mol/L mazindol or 10 kmol/L ( - ) cocaine was coapplied with the MPP+. MPP+ failed to enhance LDH release from cells or to reduce binding of C3H)CGP 1217 1, a beta-adrenergic receptor ligand, in parallel COS cell cultures transfected with pCDMP, a beta-adrenergic receptor expression plasmid @} (Fig 2C). Several steps necessary for the dopaminergic toxicity of the parkinsonism-inducing neurotoxin MPTP have been investigated as candidate mechanisms in the pathogenesis of idiopathic parkinsonism. Although controversial, current data may indicate that monoamine oxidase B ( M A 0 B) inhibition may slow the rate of progression of motor symptoms in idiopathic Parkinson's disease 143. These observations could still leave the transporter as an important participant in processes underlying idiopathic Parkinson's disease, because most human substantia nigra M A 0 B lies outside of the dopaminergic neurons [ S } . The charged nature of many M A 0 B metabolites also suggests that transport would be necessary for full passage across lipophilic neuronal membranes. Dopamine transport thus appears to deserve investigation in at least as intensive a manner as that directed toward other steps, such as mitochondrial electron transport chain activity [b, 71, for better understanding of parkinsonian pathogenesis.
A. MPP '-induced Increase in LDH Release: DAT-Transfected 0
25
MPP++ Mazlndol
50
75
100
150
125
175
200
t
-
B. DopamineTransporter Bindlng: DAT-Transfected 0
MPP'
Cocaine
20
40
60
80
100
120
*
4
C. &Receptor Binding: BAR-Transfected 0
20
40
60
80
100
120
I " " " " " "
Cocalne
Fig 2. MPP+ toxicity in COS cells transjicted with pcDNAD A T l . (A}Lactic dehydrogenase (LDH) activity in pcDNADAT1 -transfected COS cell culture supernatantsfollowing treatments with MPP+ alone or in combination with 0.1 pnzollL mazindol. Values are expressed as the percent increase over control levels and represent mean 5 SEM (n = 3). LDH release in control was 24.6 k 1.3 % of total (plate and medium) activity (287 r+ 26 mIUlsample). Asterisk indicates p < 0.05 versus control; dagger indicates p < 0.05 versus MPP+ only. (B,C) {3H}CFT and {3H}CGP12177 binding to membranes of pcDNADATl- or pCDMptransfected COS cells treated with 1 pnollL MPP+ alone or in combination with 10 pnzollL (-) cocaine or 100 pmollL isoproterenol. Values are m a n & SEM (n = 3), expressed as percent control levels. t3H}CFT and t3H}CGP12177 spec$cally bound in control were 2.25 and 20.9 fmolldish, respectively. Asterisk indicates p < 0.05 versus control.
We gratefully asknowkdge Carol Sneeringer for careful assistance with the manuscript, Chien-LiangLin for assistance with cell culture, and support from the intramural program of the National Institute on Drug Abuse and from the National Institutes of Health.
References 1. Iversen LL.. Role of transmitter uptake mechanisms in synaptic neurotransmission. Br J Pharmacol 1971;41:57 1-591 2. Horn AS. Dopamine uptake: a review of progress in the last decade. Prog Neurobiol 1990;34:387-400 3. Snyder SH, D'Amato RJ. MFTP: a neurotoxin relevant to the pathophysiology of Parkinson's disease. Neurology 1986;36: 250-258 4. The Parkinson Study Group. Effect of deprenyl on the progression of disability in early Parkinson's disease. N Engl J Med 1989;321:1364- 137 1 5. Uhl GR, Javitch JA, Snyder SH. Normal MPTP binding in parkinsonian substantia nigra: evidence for extraneuronal toxin conversion in human brain. Lancet 1985;1:956-957 6. Boyson SJ. Parkinson's disease and the electron transport chain. Ann Neurol 1991;30:330-331 7. ShoffnerJM, Watts RL,Juncos JL, et al. Mitochondrial oxidative phosphorylation defects in Parkinson's disease. Ann Neurol 1991;30:332-339 8. Shimada S , Kitayama S , Lin C-L, et al. Cloning and expression of a cocaine-sensitive dopamine transporter complementary DNA. Science 1991;254:576-5 78 9. SchaefferJC, Lin C-L, Kitayama S , Uhl GR. Ligand autoradiographic receptor screening. 11. Expression of receptor cDNA in transfected COS cells grown on polyester disks and its recovery. Mol Brain Res 1991;9:271-276 10. Cabaud PG, Wroblewski F. Colorimetric measurement of lactic dehydrogenase activity of body fluids. Am J Clin Pathol 1958; 30:234 11. Staehelin M, Simons P, Jaeggi K, Wigger N. CGP-12177: A hydrophilic P-adrenergic receptor radiohgand reveals high affinity binding of agonists to intact cells. J Biol Chem 1983; 2583496-3502 12. Madras BK, Spealman RD, Fahey MA, et al. Cocaine receptors labeled by [3H]2P-carbomethoxy-3~-(4-fiuorophenyl) tropane. Mol Pharmacol 1989;36:5 18-524 13. BojaJW, Carroll FI, Rahman MA, et al. New potent cocaine analogs: k a n d binding and transporr studies in rat striaturn. Eur J P h m a c o l 1990;184:329-332 14. Javitch JJ, DAmato RJ, Stritunatter SM, Snyder SH. Parkinsonism-inducing neurotoxin, N-methyl-4-phenyl-l,2,3,6-tetrahydropyridine: uptake of the metabolite N-methyl-4-phenylpyridine by dopamine neurons explains selective toxicity. Proc Natl Acad Sci USA 1985;82:2173-2177
The dopamine transporter can thus have a key role in mediating MPP+ toxicity. The cocaine- and mazindol-blockable toxicity of MPP' for expressing COS cells highlights the potential importance of dopamine transport for parkinsonism-inducing neurotoxic mechanisms [3].
Brief Communication: Kitayama et ak DA Transporter c D N A and MPP'
111
References 1. Barth DS, Sutherling WW, Engel JJ, Beatty J. Neuromagnetic localization of epileptiform spike activity in the human brain. Science 1982;2 18:891-894 2. Modena I, Ricci GB, Barbanera S, et al. Biomagnetic measurements of spontaneous brain activity in epileptic patients. Electroencephalogr Clin Neurophysiol 1982;54:622-628 3. Rose DF, Smith PD, Sat0 S. Magnetoencephalography and epilepsy research. Science 1987;238:329-3 3 5 4. Sutherling WW, Barth DS. Neocortical propagation in temporal lobe spike foci on magnetoencephalography and electroencephalography. Ann Neurol 1989;25 :3 73-38 1 5. Ricci GB, Romani GL, Salustri C, et al. Study of focal epilepsy by multichannel neuromagnetic measurements. Electroencephalogr Clin Neurophysiol 1987;66:358-368 6. Sutherling WW, Crandall PH, Engel JJ, et al. The magnetic field of complex partial seizures agrees with intracranial localizations. Ann Neurol 1987;21:548-558 7. Stefan H, Schneider S, Abraham-Fuchs K, et al. Application of a multichannel MEG system in temporal lobe epilepsy. In: Williamson SJ, Hoke M, Stroink G, Kotani M, eds. Advances in biomagnetism. New York Plenum, 1989:279-282 8. Tiihonen J, Hari R, Kajola M, et al. Localization of epileptic foci using a large-area magnetometer and functional brain anatomy. Ann Neurol 1990;27:283-290 9. Ahonen A, HimAilEnen M, Kajola M, et al. Multichannel SQUID systems for brain research. IEEE Trans Magn 1991; 27:2786-2792 10. Knuutila J, Ahlfors S, Ahonen A, et al. A large-area low-noise seven-channel DC SQUID magnetometer for brain research. Rev Sci Instr 1987;58:2145-2156 11. Hari R. The neuromagnetic method in the study of the human auditory cortex. In: Grandori F, Hoke M, Romani GL, eds. Auditory evoked magnetic fields and potentials. Advances in audiology. Base1 Karger, 1990:222-282 12 Williamson SJ, Kaufman L. Biomagnetism. J Magn Magn Mater 1981;22:129-202 13. Hari R, Lounasmaa OV. Recording and interpretation of cerebral magnetic fields. Science 1989;244:432-436
Parkmsonism-inducing Neurotoxin MPP': Uptake and Toxicity in Nonneuronal COS -Cells Expressing Dopamine Transporter cDNA S. Kitayama, DDS, PhD, S. Shimada, MD, and G. R. Uhl, MD, PhD
Expression of a cloned dopamine transporter complementary DNA in COS cells allows these primate kidney cells to accumulate the parkinsonism-inducing neurotoxin metabolite MPP+ (1-methyl-4-phenylpyridinium) avidly, and MPP+ toxicity results. By documenting that the dopamine transporter can confer MPP+ sensitivity to nonneural cells, these results highlight the key role that this transporter could play in mechanisms underlying parkinsonism. Kitayama S, Shimada S, Uhl GR. Parkinsonisminducing neurotoxin MPP+: uptake and toxicity in nonneuronal COS cells expressing dopamine transporter cDNA. Ann Neurol 1992;32:109-111
The dopamine transporter normally acts to terminate dopaminergic neurotransmission by sodium-dependent reaccumulation of dopamine into presynaptic neurons [l, 21.The transporter can also accumulate neurotoxins with structural features resembling dopamine; its ability to concentrate the parkinsonism-inducing toxin 1-methyl-4-phenylpyridinium (MPP' ) is required for N-methyl-4-phenyl-l,2,5,6-tetrahydropyridine (MPTP) neurotoxicity in vivo and in vitro, although other steps may also be involved [3-71. We recently cloned a functional complementary DNA (cDNA) encoding a dopamine transporter from a rat midbrain cDNA library using D N A homology and expression approaches [sf.DATl encodes a protein that can transport [3H}dopamine (E3H1DA) into Xenopus oocytes and COS cells and bind the cocaine analog { 3H]2p-carbmethoxy-3~-(4-fluorophenyl)-trope (r3H}CFT)with a pharmacological profile similar to From the Laboratory of Molecular Neurobiology, Addiction Research Center/National Institute on Drug Abuse, and the Departments of Neurology and Neuroscience, Johns Hopkins IJniversity School of Medicine, Baltimore, MD.
Received Nov 8, 1991, and in revised form Jan 7, 1992. Accepted for publication Jan 7, 1992. Address correspondence to Dr Uhl, Laboratory of Molecular Neurobiology, Addiction Research CenterlNational Institute on Drug Abuse, PO Box 5180, Baltimore, MD 21224.
Brief Communication: Kitayama et
al:
DA Transporter cDNA and MPP'
103
that of the native rat striatal transporter (81. Availability of this cDNA subcloned into a eukaryotic cell expression vector, pcDNAI (pcDNADAT I), allows assessment of the ability of this transporter to accumulate MPP+ into nonneural cells and of its ability to make accumulated MPP+ toxic to such cells.
70 _.
.
3H-DA pDATI control 3H-DA pDATl + IOpM (-) Cocaine
A 3H-DA pCDM8
0 3H-MPP+pDAT1 wntrol 3H-MPP+pDAT1 + lOpM (-1 C o ~ l l m , A 3H-MPP+pCDM8
Materials and Methods COS cells were transfected by electroporation with 20 pg/ lo7cells of pcDNADAT1, pCDM8, or pCDMbeta, as previously described IS, 91. Transfected COS cells were plated in 35-mm dishes in 2 mL Dulbecco's modified minimal essential medium containing 10% fetal bovine serum. Cells were either (1) cultured for 2 to 3 days in control medium, (2) grown in normal medium for 24 hours and changed to medium containing test drugs for 15 to 20 hours before harvesting, or (3) changed with normal medium 24 hours after transfection, to provide a control for (2). To assess neurotransmitter and neurotoxin uptake, cells were washed three times in Krebs-Ringer-HEPES (KRH) buffer at room temperature and incubated with 5 nmol/L C3H]MPPC (83.9 Ci/mmol, NEN [Dupont, Boston, MA)) or 10 nmol/L E3H]DA (50.0 Ci/mmol, Amersham, UK) in KRH buffer at 37°C. Uptake was terminated by three washes with ice-cold KRH, and radioactivity was extracted with 2 N NaOH and measured by scintillation counting. To test involvement of extracellular Na' in transport, NaCI in KRH was replaced by LiCl in some experiments. To assess MPP+ toxicity, cells and medium were assayed for: (1) lactic dehydrogenase (LDH) activity using a colorimetric procedure {lo]. Values presented represent the percent by which normalized LDH values released into the medium increased as a fraction of total (plate plus medium) LDH values. (2) r3H]CGP12177 (59 Ci/mmol, NEN) binding to beta-adrenergic receptors as previously described [ 1I]; and (3) C3H]CFT binding to dopamine transporters [l2, 13). Binding of 1 nmol/L 13H)CFT (82.7 Cilmmol, NEN) to intact cell membranes was assessed by incubation at 4°C for 2 hours in 1 mL KRH, three washes with 1 mL ice-cold buffer, extraction of radioactivity with NaOH, and scintillation counting. Nonspecific binding was defined as binding in the presence of 30 kmol/L { - ) cocaine. Statistical analyses were performed using Student's t-test.
Results and Discussion COS cells transfected with pcDNADATl displayed avid uptake of r3H}DA and E3H}MPP+ (Fig 1). Dopamine and MPP+ uptakes were rapid, sodiumand temperature-dependent, and cocaine-blockable [1-3,8} although E3H]DA uptake reached peak values more rapidly than I3H)MPP+ accumulation. COS cells do not accumulate the neurotoxin when transfected with control plasmids (see Fig 1). Expression of DATl can thus specifically confer MPP+ uptake on nonneural cells. Uptake displays interesting features. The failure of MPP+ uptake to saturate when DA transport has reached plateau values could point to differences in the intracellular sequestration or binding of the accumu110 Annals of Neurology
Vol 32 No I July 1992
0
5
10
15
20
Time (mln)
Fig 1. Time-course of f3H)MPP+ and t3H)DA uptake by COS cells transfected with pcDNADATl or vector only. Values are the means of triplicate determinations.
lated MPP', or to differences in its affinity for one of the states or forms of the transporter. Although such differences have not been clearly reported in synaptosomal studies [but see 141, these different properties of the two substrates, DA and MPP', leave open the possibility that pharmacological strategies for altering transporter function to selectively inhibit neurotoxin uptake but spare DA transport might be found. Expression of DATl conferred MPP' toxicity on transfected COS cells. Fifteen to 20 hours' exposure to lp,mol/L MPP+ increased LDH release and decreased (3H7CFT binding in pcDNADATl-expressing cultures (Fig 2A,B). Changes in each of these two markers were almost absent when 0.1 p,mol/L mazindol or 10 kmol/L ( - ) cocaine was coapplied with the MPP+. MPP+ failed to enhance LDH release from cells or to reduce binding of C3H)CGP 1217 1, a beta-adrenergic receptor ligand, in parallel COS cell cultures transfected with pCDMP, a beta-adrenergic receptor expression plasmid @} (Fig 2C). Several steps necessary for the dopaminergic toxicity of the parkinsonism-inducing neurotoxin MPTP have been investigated as candidate mechanisms in the pathogenesis of idiopathic parkinsonism. Although controversial, current data may indicate that monoamine oxidase B ( M A 0 B) inhibition may slow the rate of progression of motor symptoms in idiopathic Parkinson's disease 143. These observations could still leave the transporter as an important participant in processes underlying idiopathic Parkinson's disease, because most human substantia nigra M A 0 B lies outside of the dopaminergic neurons [ S } . The charged nature of many M A 0 B metabolites also suggests that transport would be necessary for full passage across lipophilic neuronal membranes. Dopamine transport thus appears to deserve investigation in at least as intensive a manner as that directed toward other steps, such as mitochondrial electron transport chain activity [b, 71, for better understanding of parkinsonian pathogenesis.
A. MPP '-induced Increase in LDH Release: DAT-Transfected 0
25
MPP++ Mazlndol
50
75
100
150
125
175
200
t
-
B. DopamineTransporter Bindlng: DAT-Transfected 0
MPP'
Cocaine
20
40
60
80
100
120
*
4
C. &Receptor Binding: BAR-Transfected 0
20
40
60
80
100
120
I " " " " " "
Cocalne
Fig 2. MPP+ toxicity in COS cells transjicted with pcDNAD A T l . (A}Lactic dehydrogenase (LDH) activity in pcDNADAT1 -transfected COS cell culture supernatantsfollowing treatments with MPP+ alone or in combination with 0.1 pnzollL mazindol. Values are expressed as the percent increase over control levels and represent mean 5 SEM (n = 3). LDH release in control was 24.6 k 1.3 % of total (plate and medium) activity (287 r+ 26 mIUlsample). Asterisk indicates p < 0.05 versus control; dagger indicates p < 0.05 versus MPP+ only. (B,C) {3H}CFT and {3H}CGP12177 binding to membranes of pcDNADATl- or pCDMptransfected COS cells treated with 1 pnollL MPP+ alone or in combination with 10 pnzollL (-) cocaine or 100 pmollL isoproterenol. Values are m a n & SEM (n = 3), expressed as percent control levels. t3H}CFT and t3H}CGP12177 spec$cally bound in control were 2.25 and 20.9 fmolldish, respectively. Asterisk indicates p < 0.05 versus control.
We gratefully asknowkdge Carol Sneeringer for careful assistance with the manuscript, Chien-LiangLin for assistance with cell culture, and support from the intramural program of the National Institute on Drug Abuse and from the National Institutes of Health.
References 1. Iversen LL.. Role of transmitter uptake mechanisms in synaptic neurotransmission. Br J Pharmacol 1971;41:57 1-591 2. Horn AS. Dopamine uptake: a review of progress in the last decade. Prog Neurobiol 1990;34:387-400 3. Snyder SH, D'Amato RJ. MFTP: a neurotoxin relevant to the pathophysiology of Parkinson's disease. Neurology 1986;36: 250-258 4. The Parkinson Study Group. Effect of deprenyl on the progression of disability in early Parkinson's disease. N Engl J Med 1989;321:1364- 137 1 5. Uhl GR, Javitch JA, Snyder SH. Normal MPTP binding in parkinsonian substantia nigra: evidence for extraneuronal toxin conversion in human brain. Lancet 1985;1:956-957 6. Boyson SJ. Parkinson's disease and the electron transport chain. Ann Neurol 1991;30:330-331 7. ShoffnerJM, Watts RL,Juncos JL, et al. Mitochondrial oxidative phosphorylation defects in Parkinson's disease. Ann Neurol 1991;30:332-339 8. Shimada S , Kitayama S , Lin C-L, et al. Cloning and expression of a cocaine-sensitive dopamine transporter complementary DNA. Science 1991;254:576-5 78 9. SchaefferJC, Lin C-L, Kitayama S , Uhl GR. Ligand autoradiographic receptor screening. 11. Expression of receptor cDNA in transfected COS cells grown on polyester disks and its recovery. Mol Brain Res 1991;9:271-276 10. Cabaud PG, Wroblewski F. Colorimetric measurement of lactic dehydrogenase activity of body fluids. Am J Clin Pathol 1958; 30:234 11. Staehelin M, Simons P, Jaeggi K, Wigger N. CGP-12177: A hydrophilic P-adrenergic receptor radiohgand reveals high affinity binding of agonists to intact cells. J Biol Chem 1983; 2583496-3502 12. Madras BK, Spealman RD, Fahey MA, et al. Cocaine receptors labeled by [3H]2P-carbomethoxy-3~-(4-fiuorophenyl) tropane. Mol Pharmacol 1989;36:5 18-524 13. BojaJW, Carroll FI, Rahman MA, et al. New potent cocaine analogs: k a n d binding and transporr studies in rat striaturn. Eur J P h m a c o l 1990;184:329-332 14. Javitch JJ, DAmato RJ, Stritunatter SM, Snyder SH. Parkinsonism-inducing neurotoxin, N-methyl-4-phenyl-l,2,3,6-tetrahydropyridine: uptake of the metabolite N-methyl-4-phenylpyridine by dopamine neurons explains selective toxicity. Proc Natl Acad Sci USA 1985;82:2173-2177
The dopamine transporter can thus have a key role in mediating MPP+ toxicity. The cocaine- and mazindol-blockable toxicity of MPP' for expressing COS cells highlights the potential importance of dopamine transport for parkinsonism-inducing neurotoxic mechanisms [3].
Brief Communication: Kitayama et ak DA Transporter c D N A and MPP'
111
