Psychostimulants and the regulation and trafficking of monoamine transporters

The process of chemical neurotransmission involves a dynamic balance between signal initiation and/or signal termination. Psychostimulants have long been known as drugs that alter both of these processes associated with monoaminergic neurotransmission. Specifically, it has been known for many years that cocaine and methylphenidate prevent signal termination by inhibition of dopamine (DA) and serotonin (5HT) transporters (DAT and SERT, respectively). In addition, amphetamine analogs such as amphetamine (AMPH), methamphetamine (METH), and methylenedioxymethamphetamine (MDMA), enhance signal initiation by causing release of DA and/or 5HT. More recently it has been discovered that psycho stimulants alter additional processes. In particular, amphetamines reduce the transport activity of DAT and/or SERT. The first part of this dissertation is focused on elucidating the mechanism whereby amphetamines decrease monoamine transport function. Results reveal that the METH- and MDMA-induced decrease in DAT and SERT activity can be prevented with NPC15437, a protein kinase C inhibitor. In addition, a novel mechanism of transporter regulation was discovered that involves the lipid second messenger, ceramide. The ceramide-induced alterations in monoamine transporter function appear distinct from those of METH, uncovering an interesting phenomenon: C2-ceramide reduces DA transported through the DAT while concurrently increasing the transport of 5HT through the DAT. This dissertation also contains the first evidence that psychostimulants alter the trafficking of synaptic vesicles. Cocaine and methylphenidate (drugs that are not neurotoxic to DA neurons) appear to influence the trafficking of vesicles in a manner somewhat opposite to the amphetamines (drugs which are neurotoxic to DA neurons). Results demonstrate that these vesicle trafficking events are specific for monoamine containing vesicles and occur at high as well as low, clinically relevant, doses. Data presented in this dissertation help to increase our understanding of neurotransmission and psychostimulant-induced alterations in neurotransmission. These data may be useful for understanding and treating various related neurological disorders.

PSYCHOSTIMULANTS AND THE REGULATION AND TRAFFICKING OF MONOAMINE TRANSPORTERS by Evan Lawrence Riddle A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Pharmacology and Toxicology University of Utah August 2003 Copyright © Evan Riddle 2003 All Rights Reserved THE UNIVERSITY OF UTAH GRADUATE SCHOOL SUPERVISORY COMMITTEE APPROVAL of a dissertation submitted by Evan Lawrence Riddle This dissertation has been read by each member of the following supervisory committee and by majority vote has been found to be satisfactory. Glen R. Hanson Matthew K. TBpham Bruce A. Bamber THE UNIVERSITY OF UTAH GRADUATE SCHOOL FINAL READING APPROVAL To the Graduate Council of the University of Utah: I have read the dissertation of Evan Lawrence Riddle In its final form and have found that (1) its format, citations, and bibliographic style are consistent and acceptable: (2) its illustrative materials including figures, tables, and charts are in place; and (3) the final manuscript is satisfactory to the supervisory committee and is ready for sucbm·{i s~i.o~nt t,o-.,j ,r Ti'.h e~~' G (,) r. :a)) d)u ate School. ( . . '-~ (,,--..... (, )r.'If,( :t~ 2.. . .~) J{~~ . .k~~;;~L v;::::..~. ,j Annette E. Fleckenstein Date Chair, Supervisory Committee Approved for the Major Department . /( /- i' • ' .. / / {.J / '. ~ J "/ 1(. ( / [ ,,' L , L( '.I" Wilfiam R. Cr9wley:/ Chair/Dean ,/ ./ Approved for the Graduate Council ~~\ s. d2~-- . ABSTRACT The process of chemical neurotransmission involves a dynamic balance between signal initiation and/or signal termination. Psychostimulants have long been known as drugs that alter both of these processes associated with monoaminergic neurotransmission. Specifically, it has been known for many years that cocaine and methylphenidate prevent signal termination by inhibition of dopamine (DA) and serotonin (SHT) transporters (DAT and SERT, respectively). In addition, amphetamine analogs such as amphetamine (AMPH), methamphetamine (METH), and methylenedioxymethamphetamine (MDMA), enhance signal initiation by causing release of DA and/or SHT. More recently it has been discovered that psychostimulants alter additional processes. In particular, amphetamines reduce the transport activity of DAT and/or SERT. The first part of this dissertation is focused on elucidating the mechanism whereby amphetamines decrease monoamine transport function. Results reveal that the METH- and MDMA-induced decrease in DAT and SERT activity can be prevented with NPC 15437, a protein kinase C inhibitor. In addition, a novel mechanism of transporter regulation was discovered that involves the lipid second messenger, ceramide. The ceramide-induced alterations in monoamine transporter function appear distinct from those of METH, uncovering an interesting phenomenon: C2-ceramide reduces DA transported through the DAT while concurrently increasing the transport of 5HT through the DAT. This dissertation also contains the first evidence that psychostimulants alter the trafficking of synaptic vesicles. Cocaine and methylphenidate (drugs that are not neurotoxic to DA neurons) appear to influence the trafficking of vesicles in a manner somewhat opposite to the amphetamines (drugs which are neurotoxic to DA neurons). Results demonstrate that these vesicle trafficking events are specific for monoamine containing vesides and occur at high as well as low, clinically relevant, doses. Data presented in this dissertation help to increase our understanding of neurotransmission and psychostimulant-induced alterations in neurotransmission. These data may be useful for understanding and treating various related neurological disorders. v TABLE OF CONTENTS ABSTRACT .......................................................................................... iv LIST OF FIGURES ................................................................................. ix Chapter 1. INTRODUCTION ................................................................................ 1 Psychostimulants ........................................................... '" .............. 4 Amphetamines ............................................................................... 5 Cocaine and Methylphenidate .............................................................. 6 Research Objectives ......................................................................... 7 References .................................................................................... 9 2. IN VITRO APPLICATION OF AMPHETAMINES DECREASES PLASMALEMMAL MONOAMINE UPTAKE THROUGH A PKC-MEDIATED MECHANISM ............................................................ 12 Introduction ................................................................................. 12 Materials and Methods ..................................................................... 14 Results ....................................................................................... 16 Discussion ................................................................................... 27 References ................................................................................... 30 3. CERAMIDE-INDUCED ALTERATIONS IN MONOAMINE TRANSPORTER FUNCTION: A COMPARISON WITH METHAMPHETAMINE ...................................................................... 32 Introduction ................................................................................. 32 Materials and Methods ..................................................................... 34 Results ....................................................................................... 37 Discussion ................................................................................... 42 References ................................................................................... 44 4. CERAMIDE PROMOTES THE TRANSPORT OF SEROTONIN THROUGH THE DOPAMINE TRANSPORTER ......................................... .47 Foreword .................................................................................... 47 lniroduction ................................................................................. 48 Materials and Methods .................................................................... .49 Results ....................................................................................... 49 Discussion ................................................................................... 51 References ................................................................................... 52 5. DIFFERENTIAL TRAFFICKING OF THE VESICULAR MONOAMINE TRANSPORTER-2 BY METHAMPHETAMINE AND COCAINE ..................... 54 Foreword .................................................................................... 54 Introducti()n ................................................................................. 56 Materials and Methods ..................................................................... 57 I~esults ....................................................................................... 58 Discussion ................................................................................... 58 References ................................................................................... 59 6. SELECTIVE TRAFFICKING OF THE VESICULAR MONOAMINE TRANSPORTER-2 CAUSED BY AMPHETAMINE AND METHYLPHENIDATE ......................................................................... 60 Introduction ................................................................................. 60 Materials and Methods ..................................................................... 61 Results ....................................................................................... 63 Discussion ................................................................................... 76 References ................................................................................... 84 7. CONCLUSiONS ................................................................................ 86 References ................................................................................... 92 Appendices A. TOLERANCE TO THE NEUROTOXIC EFFECTS OF METHAMPHETAMINE IN YOUNG RATS .............................................. 94 B. METHAMPHETAMINE-INDUCED RAPID AND REVERSIBLE CHANGES IN DOPAMINE TRANSPORTER FUNCTION: AN IN VITRO MODEL ..................................................................... Ion C. METHYLENEDIOXYMETHAMPHETAMINE DECREASES PLASMALEMMAL AND VESICULAR DOPAMINE TRANSPORT: MECHANISMS AND IMPLICATIONS FOR NEUROTOXICITy .................. 108 vii D. METHYLPHENIDATE REDISTRIBUTES VESICULAR MONOAMINE TRANSPORTER-2 IMMUNOREACTIVITY: ROLE OF DOPAMINE RECEPTORS .................................................... 117 E. METHYLPHENIDATE ALTERS VESICULAR MONOAMINE TRANSPORTER AND PREVENTS METHAMPHET AMINE-INDUCED DOPAMINERGIC DEFICITS ................................................ J 24 viii LIST OF FIGURES Fi gures Page 1.1 Sinlplified schematic of chenlical neurotransmission and a synaptosome ........... 2 2.1 Effects of METH on hippocanlpal synaptosomal CHJ5HT uptake .................. 17 2.2 In vitro effects of various METH concentrations on synaptosonlal [3H'J5HT uptake ............................................................................. 19 2.3 In vitro effects of METH after various times on synaptosomal ['~HJ5HT uptake ............................................................................. 21 2.4 Effects ofNPC15437 pretreatment on the METH-induced decrease In synaptosomal CHJ5HT uptake ........................................................ 23 2.5 Effects of removal of extracellular Ca++ on the METH-induced decrease in synaptosomal [,c;H]5HT uptake ............................................. 25 3.1 C2-Ceramide decreased DA uptake in a concentration-dependent manner in vitro ............................................................................. 38 3.2 Effects of C2-ceramide on CH]DA uptake in striata] synaptosomes prepared from METH-treated tissue ..................................................... 40 6.1 A single high-dose administration of MPD altered VMA T-2 subcellular localization .................................................................... 64 6.2 A single high-dose administration of AMPH altered VMAT-2 subcellular localization .................................................................... 66 6.3 A single Jow-dose administration ofMPD altered VMAT-2 subcellular localization .................................................................... 68 6.4 A single low-dose administration of AMPH altered VMAT-2 subcellular localization .................................................................... 70 6.5 A single low-dose administration of MPD rapidly and reversibly increased VMAT-2 immunoreactivity in the S3 (cytoplasmic) fraction ......... , .72 6.6 A single low-dose administration of AMPH rapidly and reversibly decreased VMAT-2 immunoreactivity in the S3 (cytoplasmic) fraction ........... 74 6.7 Phosphatase inhibition increased the phosphorylation of synapsin but did not alter VMAT-2 redistribution in the P3 (synaptosomal membrane) or S3 (cytoplasmic) fraction ............................................................... 79 x CIIAPTER 1 INTRODUCTION The functional unit of the nervous system is the neuron, which is a cell specialized for communication (Le., sending and receiving signals). These cells communicate primarily through the release of chemicals called neurotransmitters. Neurons contact and communicate with each other through structures called synapses. Communication occurs when a presynaptic neuron containing neurotransmitter stored in synaptic vesicles releases neurotransmitter into the synapse, which sequentially illicits a response in a postsynaptic neuron by interacting with receptors specialized to bind neurotransmitters (see Figure 1.1). Termination of the signaling by the neurotransmitters dopamine (DA) and serotonin (5HT) is by the re-uptake of DA or 5HT into the presynaptic neuron by a plasmalemmal DA or 5HT transporter (DAT or SERT, respecti vel y). In order for neurotransmission to continue, the DA and 5HT that is transported back into the presynaptic neuron must be packaged into synaptic vesicles for subsequent release. In the nervous system, this packaging is mediated by the vesicular monoamine transporter-2 (VMAT-2). VMAT-2 utilizes a H+ electrochemical gradient (Henry et aI., 1994) as the driving force to sequester DA and 5HT into synaptic vesicles and upon 2 Figure 1.1. Simplified schematic of chemical neurotransmission and a synaptosome. Presynaptic neuron Synapse ,......... Synaptosome ~ 4 sequestration, DA and 5HT can be released again into the synapse by fusion of the synaptic vesicles at the nerve terminal. This entire process of neuronal cell to cell communication is known as chemical neurotransmission. Psychostimulants are drugs that have profound effects on chemical neurotransmission of DA and 5HT neurons. Although much is known regarding the psychostimulant-induced changes in dopaminergic and serotonergic neurotransmission, our understanding is not complete. This dissertation describes research that investigates the mechanisms of psychostimulant-induced regulation and trafficking of plasmalemmal and vesicular monoamine transporters. Psychostimulants Psychostimulants are drugs that produce excitement, euphoria, and an increase in motor activity while lessening the sensitivity to fatigue. As a result, psychostimulants are often abused and use can lead to sensitization, tolerance (Appendix A), and addiction. Although there is abuse potential, psychostimulants are effectively used clinically for diseases such as narcolepsy, obesity, and attention deficit hyperactivity disorder (ADHD). As noted above, the exact mechanisms whereby psychostimulants alter either phsysiological or pathophysiological processes are not known, although it is well accepted that psychostimulants increase synaptic concentrations of DA and/or SHT. Psychostimulants increase synaptic concentrations of DA or 5HT through at least 2 different mechanisms: I) blocking the re-uptake of DA or 5HT into the presynaptic neuron by inhibition of DAT or SERT, or 2) increasing the release of DA or 5HT from 5 the presynaptic neuron. The psychostimulants that are described in this dissertation include amphetanlines, cocaine, and methylphenidate. Amphetamines The amphetamines are drugs that include amphetamine, methamphetamine (METH), and methylenedioxymethamphetanline (MDMA, ecstasy). These drugs are widely abused for their stimulant and euphoric effects. Abuse of amphetamines is on the rise: between 1993 and 1999, amphetamine treatment admission rates increased by at least 250 percent in 14 states (http://www.samhsa.gov/oas/2kllSpeed/Speed.cfm). One particularly dangerous amphetamine analog, METH, is widely available and access is difficult to control because it can be easily synthesized in small clandestine laboratories from household items, with the cold medicine ingredient, (pseudo )ephedrine, as the precursor reagent. It is well established that high-dose METH administration produces long-term deficits in DAT and SERT activity as well as the DA and 5HT synthesizing enzymes in rodent, non-human primates, and presumably humans (Koda and Gibb 1973; Hotchkiss and Gibb, 1980; Ricaurte et aI., 1980; Wagner et aI., 1980, Kokoshka et aI., 1998). Because of the availability, widespread and increasing abuse, and neurotoxic potential, understanding the effects of amphetamines on the brain is important. It has been known for many years that amphetamines are indirect agonists to DA and 5HT systems. As noted above, amphetamines increase extracellular DA and 5HT levels. This is achieved by enhancing DA and 5HT release (Raiteri et aI., 1975; Arnold et aI., 1977), inhibiting uptake (Harris and Baldessarini, 1973; Taylor and Ho, 1978), by 6 disrupting vesicular storage (Sulzer et aI., J 995), and inhibiting monoamine oxidase (Cho and Segal, 1994). To date there has been little study of the in vivo effects of amphetamines on YMAT-2. Chapters 2 and 3 of this dissertation focus on understanding the mechanism(s) whereby METH alters OAT and SERT activity while Chapters 5 and 6 focus on the effects of amphetamines on YMA T -2. Cocaine and Methylphenidate Cocaine is a powerfully addictive psychostimulant. Once addicted to cocaine, use becomes uncontrolled. This is particularly troubling knowing that approximately 10'*) of all 12 graders have used this drug (http://www.drugabuse.gov/lnfofax/cocaine.html). Methylphenidate (MPO) is one of the most commonly prescribed psychostimulants in the United States. Its primary clinical use is for the treatment of AOHO (Challman and Lipsky, 2000), which is estimated to effect 3-5% of children in the United States (Pincus et aI., 1995). There has been an increase in the illicit use of this drug presunlably attributable to its pharmacological similarity to other drugs of abuse, such as cocaine, and an increase in access .. Cocaine and MPO are psychostimulants that block the uptake of OA and/or 5HT (Harris and Baldessarini, 1973; Taylor and Ho, 1978). This blockade of OAT and SERT leads to increased synaptic concentrations of OA and 5HT, producing the reinforcing properties of these drugs. To date, there has been little study of the effects of these agents on VMAT-2. Chapters 5 and 6 address this issue. 7 Research Objectives As noted above~ one of the known mechanisms of action of the amphetamines is to decrease the transport activity of OAT and SERT. High-dose administration of amphetamine~ METH~ or MOMA rapidly decreases OAT and SERT activity (Fleckenstein et aI.. 1999); perhaps through events involving phosphorylation. It has been well established that incubation of synaptosomes (see Figure 1.1) or transporter­expressing cells with PMA (12-myristate 13-acetate; PCK acti vator) leads to a phosphorylation of the OAT and SERT and a decrease in transporter activity (Huff et aI., 1997; Ramamoorthy et al.~ 1999). In addition, evidence suggests that amphetamine and MOMA may activate PKC (Giambalvo, 1992a,b; Kramer et aI.. 1998). Chapter 2 of this dissertation focuses on testing the hypothesis that the METH­and MOMA-induced decrease in OAT and SERT function is due to a PKC-mediated phosphorylation of the transporter. With the use of an in vitro model system, we demonstrate that the METH- and MOMA-induced decrease in transporter activity is attenuated with a PKC inhibitor. Evidence presented in Chapter 2 supports the hypothesis that METH induces a direct phosphorylation of the OAT and SERT. Accordingly, dephosphorylation of the OAT or SERT would predictably reverse the METH-induced decrease in transporter function. Hence, Chapters 3 and 4 characterize the effects of a phosphatase activator. C2-ceramide, on OAT and SERT function. Results reveal that ceramide-induced alterations in OAT and SERT function appear to be distinct from the METH effects. Additionally. exciting results demonstrate that C2-ceramide alters the substrate specificity of OAT in that it preferentially transports 5HT (Chapter 4). 8 In Chapters 5 and 6, studies are reported on the effects of amphetamines, cocaine, and MPD on the trafficking of synaptic vesicles. The rationale for these studies come from previous findings that cocaine and alnphetamines alter the uptake of DA by the vesicular monoamine transporter-2 (VMAT-2~ Brown et aI., 2000~ Brown et at, 200 I; Appendix C) and the phosphorylation of the vesicle trafficking protein, synapsin (Smith et ai., 1993; Iwata et aI., 1996). Results reveal that anlphetamines, cocaine, and MPD differentially traffic VMAT -2-containing synaptic vesicles. However, no evidence of a role for synapsin phosphorylation was observed. The studies presented in this dissertation contribute to our understanding of neurotransmission and the impact of psychostimulants. These studies demonstrate that psychostimulants influence both the function of the plasmalemmal transporters, DAT and SERT, while also altering the subcellular distribution of the vesicular transporter, YMA T -2. Elucidation of the processes involved in neurotransmission and the mechanisms by which psychostilllulants alter transporter neurotransmission, may help improve our therapeutic strategies for treating neurological diseases. 9 References Arnold EB, Molinoff PB, Rutledge CO (1977) The release of endogenous norepinephrine and dopamine from cerebral cortex by amphetamine. J Pharrn Exp Ther 202:544-557. Brown JM, Hanson GR, Fleckenstein AE (2000) Methamphetamine rapidly decreases vesicular dopamine uptake. J Neurochem 74:2221-2223. Brown JM, Hanson GR, Fleckenstein AE (2001) Regulation of the vesicular monoamine transporter-2: A novel mechanism for cocaine and other psychostimulants. J Pharmacol Exper Tiler 296:762-767. Challman TO, Lipsky JJ (2000) Methylphenidate: its pharmacology and uses. Mayo Clin Proc 75:711-721. Cho AK, Segal OS. (1994) Amphetamine and its Analogs pp 81 1] 3, Academic Press Inc., San Diego, CA, Facts and Comparisons (2000) eNS Stimulants: Amphetamines pp. 770-772, Facts and Comparisons, St. Louis, MO. Fleckenstein AE, Haughey HM, Metzger RR, Kokoshka JM, Riddle EL, Hanson JE, Gibb JW, Hanson GR (1999) Differential effects of psychostimulants and related agents on dopaminergic and serotonergic transporter function. Eur J Pharmaco/ 382:45-49. Giambalvo C (1992a) Protein kinase C and dopamine transport-I. Effects of amphetamine in vivo. Neuropharmacology 31:1201-1210. Giambalvo C (1992b) Protein kinase C and dopamine transport-2. Effects of amphetanline in vitro. Neuropharmacology 31:1211-1222. Harris JE, Baldessarini RJ (1973) Uptake of [3HJ-catecholamines by homogenates of rat corpus striatum and cerebral cortex: effects of amphetamine analogues. Neuropharmacology 12:669-679. Henry JP, Botton D, Sagne C, Isambert MF, Desnos C, Blanchard V, Raisman-Vozari R, Krejci E, Massoulie J, Gasnier B (1994) Biochemistry and molecular biology of the vesicular monoamine transporter from chromaffin granules. J Exp BioI 196:251-262. Hotchkiss AJ, Gibb JW (1980) Long-term effects of mUltiple doses of methanlphetnlaine on tryptophar hydroxylase and tyrosine hydroxylase activity in rat brain. J Pharm Etper Ther 214:257-262. 10 Huff RA, Vaughan RA, Kuhar MJ, UhI GR (1997) Phorbol esters increase dopamine transporter phosphorylation and decrease transport V max' J Neurochem 68:225- 232. Iwata S, Hewlett GH, Ferrell ST, Czernik AJ, Meiri KF, Gnegy ME (1996) Increased in vivo phosphorylation state of neuromodulin and synapsin I in striatum from rats treated with repeated amphetamine .. J Pharmacol E~r:p Ther 278: 1428-1434. Koda L Y, Gibb JW (1973) Adrenal and striatal tyrosine hydroxylase activity after methamphetamine. J Pharm Exp Ther 185:42-48. Kokoshka JM, Metzger RR, Wilkins DG, Gibb JW, Hanson GR, Fleckenstein AE (1998) Methamphetamine treatment rapidly inhibits serotonin, but not glutamate, transporters in rat brain. Brain Res 799:78-83. Kramer KH, Poblete JC, and Azmitia EC (] 998) Characterization of the translocation of protein kinase C (PKC) by 3,4-methylenedioxymethamphetamine (MDMA/ecstasy) in synaptosomes: evidence for a presynaptic localization involving the serotonin transporter (SERT). Neuropsychopharmacology 19:265- 277. Pincus HA, Wise T, First MB (1995) Diagnostic and statistical manual (~f mental disorders, primary care version, Ed 4, pp 182-184, Washington DC; American Psychiatric Association. Raiteri M, Bertollini A, Angelini F, Levi G (1975) d-Amphetamine as a releaser or reuptake inhibitor of biogenic amines in synaptosomes. Eur J Pharmacol 34: 189-195. Ramamoorthy S, Blakely RD (1999) Phosphorylation and sequestration of serotonin transporters differentially modulated by psychostimulants. Science 285:763-766. Ricaurte GA, Schuster CR, Seiden LS (1980) Long-term effects of repeated methylamphetamine administration on dopamine and serotonin neurons in rat brain: a regional study. Brain Res 193: 153-163. Smith DA, Browning M, Dunwiddie TV (1993) Cocaine inhibits hippocampal long-term potentiation. Brain Res 16:259-265. Sulzer D, Chen TK, Lau YY, Kristensen H, Rayport S, Ewing A (1995) Amphetamine redistributes dopamine from synaptic vesicles to the cytosol and promotes reverse transport. J Neurosci 15:4102-4108. Taylor D, Ho BT (J 978) Comparison of inhibition of monoamine uptake by cocaine, methylphenidate and amphetamine. Res Commun Chem Pathol Phannacol 21:67-75. 1 I Wagner GC, Ricaurte GA, Seiden LS, Schuster CR, Miller RJ, Westley J (1980) Long­lasting depletions of striatal dopamine and loss of dopamine uptake sites fol1owing repeated administration of methamphetamine. Brain Res 181: 151-160. Introduction CHAPTER 2 IN VITRO APPLICATION OF AMPHETAMINES DECREASES PLASMALEMMAL MONOAMINE UPTAKE THROUGH A PKC-MEDIATED lVIECHANISM It has been hypothesized that amphetamines regulate monoamine transporters by protein kinase C (PKC)-mediated transporter phosphorylation (Haughey et aI., 2000, Saunders et aI., 2000). Once phosphorylated, the monoamine transporter nlay then be internalized leading to an apparent decrease in its function. Considerable evidence supports this hypothesis. For instance, Blakely and coworkers (Qian et aI., 1997; Ramamoorthy and Blakely 1999) have demonstrated in a human ernbryonic kidney (HEK-293) cell model system that the serotonin (SHT) transporter (SERT) is a phosphoprotein whose phosphorylation state is likely tightly controlled by multiple kinase and phosphatase pathways that may also influence transporter trafficking. In addition, they reported that PKC activation by phorbol 12-myristate I3-acetate (PMA) increased phosphorylation and internalization of SERT producing a decrease in serotonin uptake. It has also been reported that the methamphetamine (METH) analogs, amphetamine (AMPH) and methylenedioxymethamphetamine (MDMA, ecstasy), induce 13 the translocation of PKC from the cytosol to the plasma membrane (Kramer et al., 1998, Giambalvo, 1992 a,b). Haughey et al. (2000) demonstrated that in vivo administration of METH decreases SERT activity; an effect not due to a direct interaction of METH with the transporter. This METH-induced decrease in transporter function was attenuated by pretreatment with a dopamine (OA) 02 receptor antagonist. Since 02 receptor activation to induces the translocation of PKC (Gordon et al., 2001), it is possible that the METH­induced increase in synaptic OA concentrations sequentially activates 02 receptors, and PKC, leading to SERT phosphorylation and a decrease in function. Our laboratory developed an in vitro model for the METH-induced decrease in DA transporter (DAT) function, measured ex vivo in synaptosomes after METH treatment (Appendix B). Specifically, we demonstrated that both METH administration in vivo and METH application in vitro decrease the Vmax of dopamine uptake in synaptosomes without changing the Km or binding of the DA T ligand, WIN35428. In addition, the METH-induced decrease in vitro was not additive with the METH-induced decrease in vivo suggesting that in the two systems METH is acting via the same mechanisms. Using this n10del of METH-induced decreases in plasmalemma) DA uptake, the in vitro METH- and MDMA-induced decrease in plasmalemmal DA uptake was attenuated with prior treatment with the PKC inhibitor, NPC15437 (Figure 7, Appendix B~ Figure 4, Appendix C). The purpose of the following studies was to develop and employ a similar model to determine if PKC also mediates the impact of METH on SERT. Results reveal that METH induces a dose- and time-dependent decrease in SERT activity. This METH-induced decrease is attenuated with PKC 14 inhibitor pretreatment. The effect of extracellular Ca++ on the METH-induced decrease was also assessed since this cation may be necessary for PKC activation (Kramer et ai., 1998). Results demonstrate that the presence of extracellular CaH was not necessary for METH to exert its effects on SERT. Materials and Methods Animals. Male Sprague-Dawley rats (270-350 g; Simonsen Laboratories, Gilroy, CA) were maintained under controlled light and temperature conditions, with food and water provided ad libitun1. Drugs were administered as indicated in the legends of the appropriate figures, and doses were calculated as the respective free bases. All procedures were conducted in accordance with National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Drugs and chemicals. (±)-METH hydrochloride was generously supplied by the National Institute on Drug Abuse (Bethesda, MD). Pargyline hydrochloride and NPC 15347 (S-2,6-Diamino-N-[[ l-oxotridecyl)-2-piperidinyl]methylJ-hexanamide dihydro-chloride) were purchased from Sigma (St. Louis, MO). [7,8-3HJDA (49 Ci/mmo]) was purchased from Amersham Pharmacia Biotech (Arlington Heights, IL). [3H]5HT uptake. Uptake of [3H]5HT was determined in synaptosomes prepared according to the method described by Kokoshka et al. (1998). Briefly, fresh hippocampaJ or striatal tissue was homogenized in cold 0.32 M sucrose and centrifuged (800 x g for 12 min; 4°C). The supernatant (S 1) was then centrifuged (22,000 x g for 15 min; 4°C), and the resulting pellet (P2) was resuspended in ice-cold modified Kreb's buffer (in mM: 126 NaCI, 4.8 KCl. 1.3 CaCI2, 16 sodium phosphate, 1.4 MgS04, II dextrose, 1 ascorbic acid, 15 pH 7.4). Assays were conducted in Kreb's buffer. Each assay tube contained synaptosomal tissue (i.e., resuspended P2 obtained from 7.5 mg of original wet weight striatal tissue) and I ""M of the monoamine oxidase inhibitor, pargyline. Nonspecific values were determined in the presence of 100 nM citalopram, a serotonin selective reuptake inhibitor. After preincubation of assay tubes for 10 min at 37°C~ assays were initiated by the addition of [3HJ5HT (5 nM final concentration). Samples were incubated at 37°C for 3 min. Samples were then filtered through Whatman GF/B filters (Brandel, Gaithersburg, MD) soaked previously in 0.05% polyethylenimine. Filters were washed rapidly three times with 3 ml of ice-cold 0.32 M sucrose using a Brandel filtering manifold. Radioactivity trapped in filters was counted using a liquid scintillation counter. Remaining resuspended P2 samples were assayed for protein concentrations according to the method of Lowry et al. (1951). In METH preincubation experiments, samples were preincubated with 10 pM METH for 30 min at 37°C. After 30 min, resuspended P2 fractions were "washed II by centrifugation (22,000 x g for 15 min; 4°C). The resulting pellet (P3) was then resuspended in ice-cold Kreb's buffer, and once again centrifuged (22,000 x g for 15 min~ 4°C) to obtain a P4 pellet that was subsequently resuspended and assayed. For PKC inhibitor experiments, synaptosomes were incubated with 10 pM NPC15437 for 5 min at 37°C prior to the addition of METH. For experiments involving the removal of extracellular Ca++, the modified Kreb's buffer was made without CaClz. Statistics. Statistical analyses were performed using an ANDY A followed by a Fisher's protected least-significant difference post hoc comparison or StudenCs t test as indicated. Differences were considered significant jf probability of error was p ~ 0.05. 16 Results Results presented in Figure 2.1 demonstrate that a single high-dose administration of METH (20 mg/kg, s.c.) rapidly (within 1 h) decreased CHJ5HT uptake, as assessed in synaptosomes prepared from the hippocampi of treated rats. Previous data demonstrated that this also occurs in striatal synaptosomes (Haughey et aI., 2(00). SpecificalJy, a single administration (15 mg/kg, s.c.) decreased striatal synaptosomal 5HT uptake by approximately 25%. Figure 2.2 indicates that as observed ex vivo, incubation of synaptosonles prepared from the striata or hippocampi of nontreated rats at 37°C for 30 min with various concentrations of METH (i.e., 1-100 pM) reduced CH]5HT uptake to a degree comparable, although slightly greater, than that observed after METH treatment in vivo, ltnportant]y, this effect was not due to residual METH as synaptosomes were washed twice prior to assay~ a procedure demonstrated to remove METH from the tissue preparation (Kokoshka et aI., 1998). Because Clausing et a1. (1995) reported amphetamine brain levels after multiple adnlinistrations of 5 mg/kg drug range from R-15 JIM, and because this concentration decreased SERT activity in vitro, a concentration of 10 pM METH was used in subsequent experiments. The decrease in L3HJ5HT uptake induced by 10 pM METH occurred within 20 min but was maximal at 30 min (Figure 2.3). At time points later than 30 min, a decrease in synaptosomal viability occuned and, as a result, the 30 min time point was selected for subsequent experiments. Figure 2.4 demonstrates that the METH-induced decrease in hippocampal rH]5HT uptake was blocked by preincubation with 10 pM of the PKC-inhibitor, NPC15437. NPC15437 per se was without effect on L3Hj5HT uptake. Figure 2.5 17 Figure 2.1. Effects of METH on hippocampaJ synaptosomal CH]5HT uptake. Rats received a single injection of METH (20 mg/kg, s.c.) or saline vehicle (l ml!]g, s.c.) and were decapitated 1 h after injection. Columns represent the means, and vertical lines arc I SEM of determinations from three independent experiments. Asterisk indicates the value for METH-treated rats that differ significantly from controls (P.:5 O'()5). No • o CI) --c- CO en 18 19 Figure 2.2. In vitro effects of various METH concentrations on synaptosomal [3H]5HT uptake. Striatal or hippocampal synaptosomes were incubated with various concentrations of METH (in IlM: 1-100) or assay buffer for 30 min at 37°C and later assayed at 37°C for the influx of [3HJ5HT. Before assaying for eHJ5HT influx, synaptosomal preparations were washed two extra times, as described in Materials and Methods. Values represent the means, and vertical lines are 1 SEM of determinations from three independent experiments. Asterisks indicate the value for METH-treated synaptosomes that differ significantly from controls (p.::; 0.05). 20 CeE::nl- :,:.s:.:.1i:.=Ws u0CO .E:.:.l C- as - -C-- -.I.I-.- 0 ll-l: en ... -: c::: • 0 0 21 Figure 2.3. In vitro effects of METH after various times on synaptosomal eH]5HT uptake. Striatal or hippocampal synaptosomes were incubated with 10 JlM METH or assay buffer for various time points (5-30 min) at 37°C and later assayed at 37°C for the influx of l3HJ5HT. Before assaying for l3H]5HT influx, synaptosomal preparations were washed two extra times, as described in Materials and Methods. Values represent the means, and vertical lines are 1 SEM of determinations from three indepen4ent experiments. Asterisks indicate the value for METH-treated synaptosomes that differ significantly from controls (p ~ 0.05). 22 -c-: E 0 C") -c-: - E 0 N -c-: E ,0. .. UJ :l E0- -c-: m E E (.) 0 .:..J.. It) 0- m -J0-:- -U.....-.).. -..0..... . • c: 0 0 23 Figure 2.4. Effects of NPC 15437 pretreatnlent on the METH-induced decrease in synaptosomal [3HJ5HT uptake. Hippocampal synaptosomes were pretreated with 10 jiM NPC 15437 for 5 min and subsequently incubated with 10 JiM METH or assay buffer for 30 min at 37°C. Before assaying for l3H]5HT influx, synaptosomal preparations were washed two extra times, as described in Materials and Methods. Columns represent the means, and vertical lines are 1 SEM of determinations from three independent experiments. Asterisk indicates the value for METH-treated synaptosomes that differ significantly from controls (p ~ 0.05). I'­M ~------------~,I~.t.) o ~------------~z~ ~--------------~ --c~-: C'G ~ ______________ ~0 o 24 25 Figure 2.S. Effects of removal of extracellular Ca++ on the METH-induced decrease in synaptosomal eH]5HT uptake. Hippocampal synaptosomes were incubated with 10 flM METH or assay buffer for 30 min at 37°C with or without Ca++ in the assay buffer. Before assaying for [3H]5HT influx, synaptosomal preparations were washed two extra times. as described in Materials and Methods. Columns represent the means, and vertical lines are 1 SEM of determinations from three independent experiments. Asterisks indicate the value for METH-treated synaptosomes that differ significantly from controls (p.;:; 0.05). ++ COJ: 01- oW z:E + ~--------------~+ CO o o ~ ______________ ~z J: I­W :E CI) --c-:: CO ~ ______________ ~w N o c:i 26 27 demonstrates that the METH-induced decrease in [3HJ5HT uptake was not influenced by the removal of extracellular CaH • The effects of chelating/depleting intracellular Ca++ on the effects of METH were tested by treating synaptosomes with BAPT A-AM or thapsigargin. However, concentrations of BAPT A-AM or thapsigargin that effectively blocked the effects of METH, reduced uptake to a similar extent as METH (data not shown). Hence, no conclusions concerning the role of intracellular Ca++ in these METH effects are possible. Discussion It has been well established that in vivo administration of amphetamine analogs decreases DA T and/or SERT activity (Wagner et aI., 1980, Kokoshka et al., 1998). However, the mechanism by which this occurs is not clear. Considerable evidence demonstrates that increased phosphorylation (via PKC activation and/or phosphatase inhibition) leads to a decrease in transporter function (Huff et al., 1997; Ramamoorthy et aL, 1999). There is also evidence demonstrating that in vitro incubation or in vivo administration of the amphetamine analog, MDMA, induces the translocation (and presumably the activation) of PKC in cortical and/or hippocampal preparations (Kramer et al., 1997, 1998). This MDMA-induced translocation of PKC was blocked by the removal of calcium from the buffer during incubation with MDMA. Data presented in this dissertation demonstrate that in vitro incubation of striatal or hippocampal synaptosomes with METH or MDMA decreases SERT activity. We also demonstrate that the METH-induced decrease in hippocampal SERT activity (Fig. 2.4) is prevented with the PKC inhibitor, NPC15437. In addition the MDMA-induced decrease 28 in striatal SERT activity (Appendix C) is prevented by the PKC inhibitors, NPC 15437 and R0317549. These results suggest that PKC is involved in the METH- and MOMA­induced decrease in SERT activity, and are consistent with previous findings that MOMA activates PKC (Kramer et a1., 1997, 1998), However, the PKC inhibitor chelerythrine did not block the METH-induced decrease in OAT activity (Appendix B). It is unclear why the PKC inhibitors, NPC 15437 and R0317549, are effective in attenuating the amphetamine-induced decreases in DAT and SERT activity whereas chelerythrine is not. Since the selectivity and potency for the 12 isoforms of PKC have not been well established for these inhibitors, it is possible that chelerythrineinhibits a different subset than NPC15437 and R0317549. Additional data presented here demonstrate that extracellular calcium may not be necessary for the METH-induced decrease, whereas Kranler et al. (1998) observed that MDMA-induced activation of PKC is dependent on extracellular calcium. This discrepancy may be due to the fact that Kramer et al. (1998) observed the effects of MDMA on synaptosomes prepared from cortical cells whereas the present study observed the effect of METH on hippocampal synaptosomes or MDMA on striatal synaptosomes. In addition, it is possible that the method used by Kramer et al. (1998) detected only a subset of PKC isoforms, and the METH- or MDMA-induced isoform(s) differ(s) in our studies. It remains to be determined how amphetamine analogs affect PKC to decrease SERT activity. For instance, PKC may be stimulated to directly phosphorylate the SERT or may contribute to a cascade of events that eventually decrease SERT activity. Evidence suggesting direct phosphorylation of the SERT by PKC include findings that: 29 1) SERT has multiple sites available for phosphorylation (Ramamoorthy et aI., 1993); and 2) direct phosphorylation of SERT decreases activity (Ramamoorthy et aI., 1998). However, seenlingly conflicting data indicate that in vitro application of amphetaIninc to SERT expressing HEK-293 cells inhibits SERT phosphorylation (Ranlamoorthy et aI., 1999), Although much remains to be discovered to explain the mechanism contributing to amphetamine analog-induced decreases in SERT activity, the results of these studies provide important information to assist in bridging the gap between our knowledge that amphetamines decrease SERT activity and PKC activation decreases SERT activity. Evidence has now been provided that PKC may be involved in the amphetamine-induced decrease in SERT activity, 30 References Clausing P, Gough B, Holson RR, Slikker Jr W, Bowyer JF (1995) Amphetamine levels in brain microdialysate, caudate/putamen, substantia nigra, and plasma after dosage that produces either behavioral or neurotoxic effects. J Pharmaco[ Exp Ther 274:614-621. Giambal vo C (1992a) Protein kinase C and dopamine transport-I. Effects of amphetamine in vivo. Neurophannacology 31:1201-1210. Giambalvo C (1992b) Protein kinase C and dopamine transport-2. Effects of amphetamine in vitro. Neuropharnlacology 31: 1211-1222. Gordon AS, Yao L, Jiang Z, Fishburn CS, Fuchs S, Diamond I (200 I) Ethanol acts synergistically with a D2 dopamine receptor agonist to cause translocation of protein kinase C. Mol Pharmacol 59: 153-160. Haughey HM, Fleckenstein AE, Metzger RR, and Hanson GR (2000) The effects of methamphetamine on serotonin transporter activity: role of dopamine and hyperthermia. J Neurochem 75:1608-1617. Kokoshka 1M, Metzger RR, Wilkins DG, Gibb JW, Hanson GR, Fleckenstein AE (1998) Methamphetamine treatment rapidly inhibits serotonin, but not glutamate, transporter in rat brain. Brain Res 799:79-83. Kramer KH, Poblete JC, and Azmitia EC (1998) Characterization of the translocation of protein kinase C (PKC) by 3,4-methylenedioxymethamphetamine (MDMA/ecstasy) in synaptosomes: evidence for a presynaptic localization involving the serotonin transporter (SERT). Neuropsychophannac%gy 19:265- 277. Qian Y, Galli A, Ramamoorthy S, Risso S, DeFelice LJ, and Blake]y RD (1997) Protein kinase C activation regulates human serotonin transporters in HEK-293 cells via altered cell surface expression. J Neurosci 17:45-57. Ramamoorthy S, Blakely RD (1999) Phosphorylation and sequestration of serotonin transporters differentially modulated by psychostimulants. Science 285:763-766. Sandoval V, Riddle EL, Ugarte YV, Hanson GR, and Fleckenstein AE (2000) Methamphetamine-induced rapid and reversible changes in dopamine transporter function: an in vitro model. J Neurosci 21:1413-1419. Saunders C, Ferrer JV, Shi L, Chen J, Merrill G, Lamb ME, Leeb-Lundberg LMF, Carvelli LC, Javitch JA, Galli A (2000) Amphetamine-induced loss of human 31 dopamine transporter activity: an internalization-dependent and cocaine-sensitive mechanism. Proc Natl Acad Sci USA 97:6850-6855. Wagner GC, Ricaurte GA, Seiden LSl Schuster CR, Miller RJ, Westley J (1980) Long­lasting depletions of striatal dopamine and loss of dopamine uptake sites following repeated administration of methamphetamine. Brain Res 181: 151-160. CHAPTER 3 CERAMIDE .. INDUCED ALTERATIONS IN MONOAMINE TRANSPORTER FUNCTION: A COMPARISON WITH METHAMPHETAMINE Introduction Recent studies demonstrated that METH administration rapidly and reversibly reduces the activity of dopamine (OA; Fleckenstein et aI., ]997a~ Kokoshka et al. 1998b) and serotonin (5HT~ Kokoshka et aI., 1998a; Haughey et aI., 2000b) transporters (OAT and SERT, respectively), as assessed in synaptosomes prepared from treated rats. These effects were not due to residual drug introduced by the original treatment since the effects persisted even when METH was "washed" from the synaptosomal preparation (Fleckenstein et aI., 1997a; Kokoshka et aI., 1998a). Norepinephrine (NE) transporter (NET) activity was also reduced, but unlike effects on OAT and SERT, this decrease was el1minated by washing the synaptosomal preparation (Haughey et aI., 2000a). OA contributes to the long-term decrease in OAT and SERT function caused by multiple METH injections, as evidenced by findings that OA depletion prior to METH treatment attenuates these METH-induced deficits (Metzger et aI., 2000; Haughey et aI., 2000b). As described in Chapter 2 and Appendices Band C of this dissertation, phosphorylation also appears to contribute to decreases in DA T function caused by METH and MDMA, 33 since pretreatment with a protein kinase C inhibitor attenuates the effects of METH on OAT and SERT in vitro. Still, much remains to be elucidated regarding mechanisms whereby this stimulant alters transporter function. Ceramides are lipid second messengers with a variety of functions (for review. see Mathias et aI., 1998). Several factors suggest that these messengers may mediate the METH-induced change in monoaminergic transporter function. For instance, ceramide generation (hydrolysis of sphingomyelin or de novo synthesis) is triggered by reactive oxygen species (ROS) formation (Verheij et aI., 1996; Singh et aI., 1998~ Mansat-De Mas et aI., 1999), and ROS production is enhanced after METH treatment (Kondo et aI., 1994~ Giovanni et aI., 1995; Fleckenstein et aI., 1997b). In addition, ceramide induces protein phosphorylation (Muller et aI., 1995; Oobrowsky and Hannnun, 1992~ Tanabe et aI., 1998~ Huwiler et aI., ]998), and phosphorylation of OAT (Vaughan et aI., 1997; Huff et a1.,1997~ Zhu et aI., 1997) and SERT (Ramamoorthy and Blakely, 1999~ Quian et aI., 1997) decreases function of these two transporters. Moreover (and as noted above), phosphorylation may contribute to the METH-induced decrease in OAT function (Appendix B; Chapter 2). Finally, ceramide causes OA release (Blochl and Sirrenberg, 1996), and, as mentioned above, OA contributes to the decrease in OAT and SERT function caused by multiple METH injections (Haughey et aI., 2000b; Metzger et aI., 2000). Hence, ceramide was evaluated as a possible candidate by which METH influences monoan1ine transporters. Specifically, the effects of direct application of C2- ceramide (a cell permeable analog of cerami de ) in vitro on synaptosomal OA uptake were tested and compared with effects resulting from METH treatment. For comparison, cerami de effects on SERT and NET activity were also determined. 34 The results revealed that C2-ceramide, like METH, profoundly decreased DA T activity~ a phenomenon that will be addressed in Chapter 4. However, in contrast to METH, C2-ceramide did not alter NE uptake and actually increased SERT function. This latter phenomenon is characterized in detail in Chapter 4. Further evidence, including direct measurement of ceramide levels after METH treatment, failed to suggest a role for ceramide in METH-induced decrease in monoamine transporter function. Still, these data provide the first demonstration that ceramide alters monoamine transport. In addition, ceramide may be among the first compounds identified to rapidly increase SERT activity. Materials and Methods Animals. Male Sprague-Dawley rats (300-350 g: Simonsen Laboratories, Gilroy, CA) were maintained under conditions of controlled temperature and lighting, with food and water provided ad libitum. Rats received METH or saline vehicle (s.c.) as indicated in the text, and all drug concentrations were calculated as free base. Animals were sacrificed by decapitation. All experiments were conducted in accordance with the National Institutes of Health guidelines. Drugs and chemicals. (±)METH hydrochloride and (-)cocaine hydrochloride were provided generously by the National Institute on Drug Abuse. Citalopram hydrochloride was supplied kindly by H. Lundbeck, pargyline hydrochloride was obtained from Abbott Laboratories (North Chicago, IL), and desipramine hydrochloride was purchased from Research Biochemicals International (Natick, MA). [7,8- 3H]Dopamine (46 Cilmmol) was purchased from Amersham Life Sciences (Arlington 35 Heights, IL). 5-[1 ,2,_3H(N) ]-hydroxytryptamine (30 Ci/mmol) and y[ 32pJ A TP (3000 Ci/nlmol) were purchased from New England Nuclear (Boston, MA). C2-ceramide and C2-dihydroceralnide were purchased from Calbiochem (San Deigo, CA). Brain ceramide was purchased from Avanti Polar Lipids (Alabaster, AL). The sn-l ,2-Diacylglycerol (DAG) assay reagents system was purchased from Amersham (Piscataway, NJ). [3H]Neurotransmitter uptake. Synaptosomal uptake of [3H]neurotransmitter was determined according to a modification of a method described by Fleckenstein et al. (1996). Striata or hippocampi were homogenized in ice-cold 0.32 M sucrose and centrifuged (800 x g for 12 min; 4°C). The supernatant (S 1) fractions were then carefully removed and centrifuged (22,000 x g for 15 min; 4°C) and the resulting pellet (P2) was resuspended in ice-cold 0.32 M sucrose. Assays were conducted in modified Kreb's buffer (in mM: 126 NaCI, 4.8 KCI, 1.3 CaCI2, 16 sodium phosphate, 1.4 MgS04, 11 dextrose, 1 ascorbic acid; pH 7.4). Transport of [3H]DA, [3H]5HT. and [3H]NE, was determined in synaptosomal tissue (i.e., resuspended P2) obtained from 1.S, 7.S, or 10 mg original wet weight of tissue per reaction tube, respectively. All reaction tubes also contained ImM pargyline. Nonspecific values of the CH]DA, CH]SHT, and [3H]NE were determined in the presence of 1 mM cocaine, 10 11M citalopram, and 10 ,...,M desipramine, respectively. Ceramide was added to the assay tubes immediately prior to the incubations. All tubes were incubated for 10 nlin at 37°C and assays were initiated by addition of O.S nM [3H]DA (except kinetic experiments wherein 0.5 nM to 10 ,...,M was used), S nM [3HJ5HT, or 5 nM [3HJNE. Samples were incubated for an additional 3 min (DA and SHT) or S min (NE) at 37°C, then filtered through Whatman GF/B fjlters soaked previously in O.OSo/cl polyethylenimine. Filters were washed rapidly three times with 3 ml 36 ice-cold 0.32 M sucrose using a Brandel filtering manifold. Radioactivity trapped in filters was counted using a liquid scintillation counter. For ceramide washout experiments, P2 pellets were resuspended mice-cold modified Kreb's buffer and incubated with 100 ~M ceramide for 15 tnin. After incubation, tubes were placed on ice and then centrifuged (22,000 x g for 15 min~ 4 DC). The supernatant was discarded and pellets were resuspended in modified Kreb's buffer containing I mM pargyline prior to preincubation and addition of eHJDA. Ceramide concentrations. Striatal tissue from saline- and METH-treated rats was homogenized in a phosphate-free buffer containing 50 tnM HEPES and 1 mM N aCI. Lipids were extracted by the method of Bligh and Dyer (1959) and then used to quantitate ceramide and total lipid phosphate. Ceramide levels were measured by a modification of the diacyJglycerol kinase assay (Okazaki et aI., 1990~ Preiss et aI., 1986) using the DAG assay system (Amersham) according to the manufacturers instructions with two modifications. First, brain ceramide (0-2500 pmol) was used for the standard curve instead of diacylglyceroL and second, lipids were separated by thin layer chronlatography using a CHCI3:methanoi:acetic acid (325:75:25) solvent system. Ceramide levels were normalized to total lipid phosphate levels determined from the lipid extracts as previously described (Whatley et aI., 1993). Data analysis. Statistical analyses between two groups were conducted by a two­tailed Student's t test. Analyses among three or more groups were conducted with analysis of variance followed by Fisher's test. Differences among groups were considered significant if the probability of error was less than 5%. 37 Results Results presented in Figure 3.1 demonstrate that incubation with C2-ceramidc produced a concentration-dependent decrease in DA T function as assessed in vitro after application to rat striatal synaptosomes. This phenomenon is characterized in detail in Chapter 4. At a concentration of 100 ~M, C2-ceramide decreased DAT function by 56%, (Figure 3.1), an effect siluilar in magnitude to a single high-dose administration of METH (Fleckenstein et aI., 1997a~ Kokoshka et al. 1998b). For this reason, 100 ~M C2- ceramide was used in the remaining experiments. Results presented in Figure 3.2 confirmed previous studies (Fleckenstein et ai., 1997a~ Kokoshka et al. 1998b) that a single METH injection (15 mg/kg, s.c.) rapidly decreases CH]DA uptake, as assessed in synaptosomes prepared from treated rats. This is approximately the maximal effect caused by a single METH administration. Ceramide application in vitro to synaptosomes prepared from rats treated with METH in vivo further decreased [3H]DA uptake. More specifically, the effects of ceramide and METH were additive, suggesting that these treatments affect uptake via different mechanisms. This conclusion was confirmed by findings that neither a single (15 mg/kg, s.c.) nor multiple (4 injections of 10 mg/kg, s.c., 2-h intervals) METH injections altered striatal ceramide levels (mean concentrations for control, single injection, and multiple injection groups were 20.46 ± 1.56,20.37 ± 1.06,18.34 ± 0.53 pmo] ceramide/nmol phosphate, respectively (p = 0.385)). In contrast to effects on DAT, C2-ceramide did not decrease NET function as assessed in hippocampal synaptosomes (values for control and ceramide-treated synaptosomes were: 6.9 ± 0.] and 7.7 ± 1.0 fmol/mg tissue (original wet weight), 38 Figure 3.1. C2-Ceramide decreased DA uptake in a concentration-dependent manner in vitro. Assays were conducted using 0.5 nM [3HJDA as described in the Methods section. Values represent means (fmol/mg tissue [original wet weight])..i" I SEM of three determinations. *Value for C2-ceramide-treated synaptosomes that is significantly different from control (p :5. 0.05). o 0 000 ~ Ct) C\J .,.... o .o,... . 39 o~ L()~ ::s ~ (I) -c 0.,...·.E-ctS '- (I) (.) I N .,....0 o (anssn6w/iOwl) alle~dn VO[H£] 40 Figure 3.2. Effects of C2-ceramide on l"H]DA uptake in striatal synaptosomes prepared from METH-treated tissue. Rats received METH (15 mg/kg s.c.) or saline vehicle (1 ml/kg s.c.) 1 h before decapitation. The data are from a single experiment and expressed as a percentage of the control. The control value for [3H]DA uptake (fmoll mg tissue [original wet weight]) was 31.3 ± 0.8. All samples were run in triplicate. 0000000000 o 0) eX) r-- CD an ~ M N ,... ~ (IOJIUOO %) a>teldn VC[H£] 0 J: I­W ~ --Cc-I) E CO 1- CI) 0 -0 .1..- s::: 0 0 41 42 respectively). Also in contrast to effects on OAT, ceramide application increased SERT function (as reported in detail in Chapter 4; values for control and ceramide-treated synaptosomes were: 19.2 ± 0.9 and 25.4 ± 1.6* fnl01/mg tissue (original wet weight: *p ~ 0.05). The inactive ceramide analog, C2-dihydroceramide, had no significant effect on 5HT uptake at a concentration of 100~M (data not shown). Discussion METH has a profound effect on monoamine transporters that contributes to its pharmacological and abuse properties. Recent studies indicate that both a single and multiple injections of METH rapidly and transiently decrease OAT and SERT function (Kokoshka et aI.. 1998a,b~ Haughey et aI., 2000a~ Fleckenstein et aI., 1997a) in a non­competitive manner. The purpose of the present study was to investigate whether ceramides contribute to these METH-induced effects. The ceramides are ubiquitous lipid second messengers that are produced as a result of the hydrolysis of sphingomyelin or by de novo synthesis. They have a variety of functions. including the ability to cause OA release (Blochl and Sirrenberg, 1996) and mediate protein phosphorylation (Muller et aI., 1995; Dobrowsky and Hannnun, 1992; Tanabe et aI., 1998: Huwiler et aI., 1998). Since OA and phosphorylation have been implicated in causing the decrease in DAT function after multiple METH injections in vivo (Metzger et aI., 2(00), and a single METH treatment in vitro (Appendix B). respectively, the effects of ceramide on DAT were assessed. Ceramide (1 00 ~M) produced similar decreases in DA uptake compared to a single METH injection. However, the present data suggest that the lipid messenger does not mediate effects of the 43 stimulant. Specifically, ceramide decreased DA T function in synaptosomes prepared fromMETH-treated rats (Figure 3.2). The fact that the effects of the ceramides were additive with the luaximal effects caused by a single METH administration, suggests that METH and ceramide have different mechanisms. In addition, METH treatment did not alter ceramide levels, further suggesting that ceramide does not mediate the METH­induced changes. However, these findings do not preclude the possibility that ceramide is locally or transiently generated within the neurons which would reduce DAT function without producing a detectable quantitative change in cellular ceramide levels. Additional evidence that ceramides do not mediate the METH-induced changes in transporter function are findings that ceramide increases 5HT uptake whereas METH decreases 5HT uptake (Haughey et aI., 2000b; Kokoshka et aI., 1998b). This exciting result demonstrates that the uptake of 5HT can be increased above basal function by physiological factors such as ceramide. This phenomenon will be discussed in Chapter 4. Noteworthy is the finding that not aU transporters are affected by ceramide treatment. For instance, NET was not affected by the second messenger. This finding, coupled with the bidirectional effects of ceramide on DA T and SERT function, underscores the differences in regulation of transporters (Chapter 4). In conclusion, C2-ceramide, like METH, decreased DA uptake into striatal synaptosomes. However, ceramide did not appear to mediate this stimulant-induced effect. In addition, ceramide increased 5HT uptake into striatal synaptosomes. 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Okazaki T, Bielawska A, Bell RM, Hannun Y A (1990) Role of ceranlide as a lipid mediator of ] n,25-dihydroxyvitamin D;1-induced HL-60 cell differentiation. J Biol Chem 265: 15823-15831. Preiss J, Loomis CR, Bishop WR, Stein R, Niedel IE, Bell RM (1986) Quantative measurement of sn-l ,2-diacylglycerols present in platelets, hepatocytes, and ras­and cis-transformed normal rat kidney cells. J Bioi Chem 261:8597-8600. Quian Y, Galli A, Ramamoorthy S, Risso S, DeFelice LJ, Blakely RD (1997) Protein kinase C activation regulates human serotonin transporters in HEK-293 cells via altered cell surface expression. J Neurosci 17:45-57. 46 Ramamoorthy S, Blakely RD (1999) PhosphoryJation and sequestration of serotonin transporters differentially moduJated by psychostimulants. Science 285:763-766. Singh 1, Pahan K. Khan M, Singh AK (1998) Cytokine-nlediated induction of ceramide production is redox-sensitive. J Bioi Chern 273:20354-20362. Tanabe F, Cui S, Ito M (1998) Ceramide promotes calpain-mediated proteolysis of protein kinase C ~ in murine polymorphonuclear leukocytes. Biochem Biophys Res Comrn 242:129-133. Vaughan RA, Huff RA, UhI GR, Kuhar MJ (1997) Protein kinase C-mediated phosphorylation and functional regulation of dopamine transporters in striatal synaptosomes. J Bioi Chern 272:15541-15546. Verheij M, Bose R, Lin XH, Yao B, Jarvis WD, Grant S, Birrer MJ, Szabo Zon LI, Kyriakis JM, Haimovitz-Freidman A, Fuks Z. Kolesnick RN (1996) Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis. Nature 380:75-79. Whatley RE, Stroud ED, Bunting M, Zimmerman GA, McIntyre TM, Prescott SM (1993) Growth-dependent changes in arachadonic acid release from endothelial cells are mediated by protein kinase C and changes in diacylglycerol. J Bioi Chem 268: 16130-16138. Zhu S, Kavanaugh MP, Sonders MS, Amara SG, Zahniser NR (1997) Activation of protein kinase C inhibits uptake, currents and binding associated with the human dopamine transporter expressed in xenopus oocytes. J Pharmacol Exp Ther 282: 1358-] 365. Foreword CHAPTER 4 CERAMIDE PROMOTES THE TRANSPORT OF SEROTONIN THROUGH THE DOPAMINE TRANSPORTER The previous chapter was designed to determine if the lipid second messenger, C2-ceramide, is inv01ved in the methamphetamine-induced decrease in monoamine transporter function. The data suggest that the ceramide-induced alterations in monoamine transporter function are not related to methamphetamine-induced decreases. However, these studies produced two interesting results: 1) C2-ceramide decreases dopamine transporter function (Figure 1), and 2) C2-ceramide increases serotonin uptake into striatal synaptosomes. This is the first report that a lipid second messenger can alter monoamine uptake. As a result, the following studies were designed to investigate the effects of C2-ceramide on dopamine and serotonin uptake. Reprinted with permission from the European Journal of Pharmacology. Available oniine at www.sciencedirect.com SCIENCIlt@DlJtIlCT" e.1P ELSEVIER European Journal of' Pharm<lcnlnl!Y 45X 120(3) 31 36 ww".clscv\cr.comilocatc,cJphar Ceran1ide-induced alterations in dopamine transporter function Evan L. Riddle'\ Kristi S. Rau'\ Matthew K. Topham b , Glen R. Hanson il , Annette E. Fleckenstein a.* '1)<,/,,,,-111/(,,,1 (If Plum",}colog) (Uullin/co/f)!!..). L!uiw!rsill' ,,(Utu!;. Room 2IJ/. 3IJ .'lOll1li ::0(10 East. Sali La;",' ("11'. L!F X4fl:!. CSI "I fllllismall ('(111"")' Ills/ilule. l'lIjwrs!/l' oj Ulah. Salt Lake (';11', L'T 114 f n. ['.',:./ RccClved III Ocwber 20()2; rccclwd In revised loml 4 Novcmhcr 2002; accepted (, Novemocr Abstract The purpose of this study was to detenninc the cl1cets oleeramide on dopamine and serotonin (5-HT, 5-hydroxytryptamine) transporters, Exposure of rdt stnatal synaptosomcs to C2-ceramide caused a reversih1c. concentration-dependent decrease in pla~ma1cmmal dopamine uptake. In contrast. cerdmide exposure increased striatal 5-HT synaptosomal uptakc. This increase did not appear to he due to an increased uptake by the 5-I1T transporter. Rather. the Increase appearcd to result from an increasc in 5-HT tmnsport through the dopamine transporter, an ;Isscrrion evidenced hy findings that this increase: (I) docs not occur in hippoemnpal synaptosomcs \ i.e .. a preparation largely devoid of dopamine transporters), (2) oceu" in striatal synaptosomes prepared from para-chloroamphetamine-treated rats (i.e .. a preparation lacking 5- HT transporters). (3) is attenuated by pretreatment with methylphcnidate (i.e .. a relatively se\cetive dopamine reuptake inhibitor) and (4) lS inhibited by exposure to exogenous dopamine (i.c .. which presumably compctes for uptake with 5-HT), Taken together. these results reveal that ceramidc is a novel modulator of monoaminc transporter function. and may alter the affinity of dopamine transporters for its primary substrate, .(' 20():! Elsevier Science H.Y. All rights reserved, ""\'wol'ds Dopamine; S-HT IS-hydroxytryptamine. serotonin) transporter; Ccrarnidc; Uopamine transporter I. Introduction The discovery of inhibition of protein kinase C by sphingosine n Ll1lnun ct ,d., 19t;6) led to the suggestion that sphingolipid-derived products act as lipid second messengers. Subsequent studies demonstrated that ceram­ide. a cleavage product of sphingomyelin, alters the phos­phorylation state of a variety of proteins. These etH:cts on phosphorylation arc undoubtedly complex, as ceramidc modi lies the activity of a variety or enzymes induding cytosolic senne'threonine protein phosphatases (Dohrmv 'k) and llannun. i'N2), protein kinase C (i:mab,: (;1 111.. i')'}>;) and stress-activated protein kinases (\Vcs,\"'id, <II .. l1N5i. or interest are recent findings that ceramide can alter the function of several distinct transporter molecules, including p-glycoproteins (for review, sec Sictsrnn aL 20U j ). ('()fTCSI10nl:!m)! author. Tel + l-~O 1-5~5· 7474; fax: + I-~() 1-585-S11 1 I1cd,cn~ICin'(l:hsc,uLlh,edl1 (A.E. Fleckenstein) Although never reported, ceramides would predictably alter monoamine transport as well, since: (I) kinases such as protein kinase C (Copd~l11J l~t :Ii.. h'96: Vaugbm~ ct aL I')()]) and PI-3 kinase (Carvel!i cl al. ::'(01) regulate dopamine uptake, and (2) phosphorylation or the dopamine (Vaughan et al.. Ii uf( e1 0.11.. 1997 : /11\1 el 19'P J and serotonin (5-HT; 5-hydroxytryptamine) transporters ((linn ,:1 aL I'):)': Ramamoorthy and Blakely. 19(j9) decreases these carriers' activities, Accordingly. the pur­pose of the present study was to detemline if ceramide alters monoamine transport. Specifically, tht: elreets or applying C2-ceramide (a eell-pemleable analog of ceram­ide) were assessed, Results reveal that C2-ceramidc pro­foundly decreases dopamine uptake into rat striatal synaptosomes. In contrast, C2-ceramidc increases synapto­somal 5-HT uptake. Interestingly, tIllS increase in :i-lIT uptake appeared to occur via the dopamine transporter. These data provide the tirst demonstration that ceramide alters monoamine transport and that a lipid second mes­senger may change the substrate-speciticity of a mono­amine transporter. 00 1 4-:!'I99:02 '$ • front matter ( 2002 Science B.Y. All rights reserved Pit son 14-29'1'/(02 )H}727-l) 48 32 F:' L RiddJ.· £'1 of . .' Europ£'all jOl/mal of Pharmac%gr 458 (2()03) 31· 36 2. Materials and methods 1. Animals and treatments Male SpmgueDawlcy rats (300350 g; Simonsen Lab­oratories, Gilroy, CAl were maintained under conditions of controlled temperature and lighting. with food and water provided ad libitum. Animals were saeritleed by decapita· tion. All experiments were conducted in accordance with the National Institutes or Health guidelines. :!.:!. Drugs and chemicals (- H_:ocaine hydrochloride and (± )-methylphenidate were provided generously by the National Institute on Drug Abuse. Para-chloroamphetamine was purchased from Sigma (St. Louis. MOl. Citalopram hydrochloride was supplied kindly by II. Lundbeck. and pargyline hydro­chloride was obtained from Abbott Laboratories (North Chicago, IL). l7.x-1H1Dopamine (46 Ciimmol) was pur­chased fhlln Amersham Life Sciences (Arlington Heights. TLl. 5-11.2,-'H(M]hydroxylryptamine (25.5 Ciimmol), IN­methyl- 1H1WIN35428 I( - )-2-i"-carbomethoxy-3-1,>-(4-fluo­rophenyl) tropane 1,5-naphthalenedisulronate; R4.5 Ci.' mmolj and 'YI'2p] ATP (3000 Ciimmol) were purchased from New England Nuclear (Boston, MAl. C2-ccramide and C2-dihydroceramide were purchased from Calbiochefll (Sail Diego, CAl, Brain ceramide was purchased from Avanti Polar Lipids (Alabaster, AL). The sn- J ,2-diacylgly­cerol assay reagents system was pllfchased from Amersham (Piscataway, NJ). .::',3. Svnaptosoma/ tHjncurotransmitter uptake and tHl IfllN.15428 binding Synaptosomal uptake of I'Hlneurotransmitter was deter­mined as described by Flcckclbkin d ~d, ( I Q')7). C2-ceram­ide was added to reaction tubes and incubated at 37°C for 10 min prior to the 3-min incubation with ['Hlneurotransmitter. I~H)WIN3542S binding experiments were perfonned as described by Kokoshka cl ,IL (199x L For ceramide washout experiments, P2 pellets were resuspended in ice-t:old modified Kreb's butler and incu­bated with I 00 ~tM ceramide fix 10 min. After incubation. tubes were placed 011 ice and then centrifuged (22,000 x g t()r 15 min: 4 'C). The supernatant was discarded and pellets were resuspended in modified Kreb's butTer containing 111M pargyline prior to preincubation and addition of (lHldop­amine. 2.4. ('{'ramide determinations Rat striatal tissue was homogenized in a phosphate-tree buffer containing 50 mM HEPES and I mM NaCI. Lipids were extracted by the method of Bligh ami Dyer (1959), and then used to quantitate ceramide and total lipid phosphate. Ceramide leveb were measured by a modification of the diacylglycerol kinase assay (Obzaki ci :IL I 99(); PfI:I:;'; cl al.. ! ()~61 using the diacylglycerol assay system (Amersham) according to the manut:lclurers instructions with Iwo mod­jfications. First, brain ccramide (0··2500 pmof! was used tor the standard curve instead of diacylglycerol, and second, lipids were separated by thin layer chromatography using a CIK'!,!methanol'acetic acid (325:75:25) solvent system, Ceramide levels were llon11alized 10 total lipid phosphate levels detennined trom the lipid extracts as previously described IWI1;ll"') ],5. Oata 1.lI1a/j·sis Statistical analyses between two groups was conducted by a two-tailed Student's t-Iest. Analyscs among three or more groups were conducted with analysis of variance followed by Fisher's test. Differences among groups were considered signi ficant if the probability of error was less than 5%" 3. Results Results presented in Fil' ! demonstrate that incubation or rat striatal synaptosomes at 37°C lor 10 min with C2- ceramide in vilro produced a concentration-dependent decrease in dopamine uptake. Concentrations or I and 10 liM had no effect, while 50 and 100 liM produced an I x~;, and 60% decrease in dopamine uptake, respectively. In contrast, incubation with the inactive C2-ceramide precursor, C2-dihydroeeramide, had no etTcct on j1H]dopamine uptake at concentrations up to 100 liM (data not shown). The ceramide-induced decrease occurred as early as 5 min and persisted lor 20 min (control I.(n 0,09, 5 min 0.42 ± O.Of(*, I () min 0.19 ± (U) I * and 20 min 0.05 0.0 I '" l'moll llg protein; n "" 3: * P 0.05). Effects of ceramide application lor greater time periods were not assessed owing to a loss of O~~~~~M~!~~~-W~ o 10 SO 60 70 80 90 100 Ceramlde (pM) Fi~ I, Synuptosomes were incubat.:d wilh C2·ceralmdc or vehicle for I () mm pnor to (hI: addition or r 'H]dopamine, Values represent means and veltieal linc., I S.E.M. oCdd':lTIlinalions i'romlhrcc independent experiments. with samples in each experiment run inlriplica\c. 'Value for C2-ccramide-In:alcd synaptosomes tltat b significantly different Crom control II' '.' 0.(5) 49 tL Riddle el <II 'European JOllrnal olPilarma{olo.fi1· 458 (lOO}) 31~3(j :[ 0.6 (5 a c:n S ~ 0,4 -:e.. ~ !c!!. :::l 0.2 « Q ~ Control Ceramide Control Wash Ceramide Wash Fi).! SYl1aptostllncs ,,-ere incuhatcd with 100 ~tM C2-ccramidc or vchide Illt 10 min prior 10 the addillon or ['I!]dopaminc. "Washed" tissue was incubated with 100 11M Cl-cammde I(l[ 10 min prior 10 wash. as described m SccUon 2. Columns represent means and vmical lincs I S.E.M. or dctertmnations from three mdependent experiments, with samples in each expenment run tn *V"luc l{)f C2-ccramide-trcated synaptosomcs thaI is signilicantly irom control ( P::: OJ)'i J. viability or the synaptosome::; (data not shown). To inves­tigate the physiological relevance of this phenomenon, ccramidc levels were assessed and determined to be present in rat striatum (mean concentrations of 20A6 1,56 pmol ceramidc'nmol phosphate), At a concentration of 100 ~IM, C2-ceramide incubation for 10 min produced a 60% decrease in 1 3 II Jdopamine up­take, eflects similar in ma!:,rnitude to application the protein kinase C-activating phorbol esters (Vaughan l'1 :11., 191.)h For this reason, 100 pM C2-ceramide was used in the remaining experiments. The ceramide-induced decrease in 11H ldopamine uptake caused by incubation with 100 JlM ceramide was attributable to a decrease in Vmas (1495.9 vs. 019,1 fmol/mg/min for control and C2-ceramide-treated synaptosomes, respectively) and no eirect on Kill (73.3 vs, 63,9 nM for control and C2-ceramide-treated synaptosomes, respectively). Moreover, both C2-ceramide and C2-dihydro- Ii) 45 ::l :l 40 ;35 E ~ 30 ~25 ~ 20 11:1 0.15 ::J 1-10 ::c .;, 5 £' * ~ 0 ~C~o-n-tr-o~1 -Ceramide 100 nM Citalopram 100 nM Cltalopram hi'. . .I. Syn~plosomes were incuhated with C2-c.:rmnide. cilalopram and/or vehicle lor 10 min pnor to the addition of ['\1J5-HT Cotumns represent means and verttcal Imes I S.E.M. of detcmltnlltlons from two ll1dcpcndcnt cxperlments. with samples in each run in triplicate. *Valuc lilr ('c-ccramldc-trcated synaptosomes significantly dilTcrcnt from control (1' OJ)5) * Vehicle ... Ceramide Saline MPD MPD Fil.!. 4. Synaptosomcs were incuhated with C2-ccramldc, methylphcnidmt: (M!'D) a~d!or vehiclc Ill[ 10 min pnor 10 the additIon 01'1 'i !!:>-flT. Columns represent means and venical lInes I S,E,M of ddcm!inatlOns frlm! three ttldcpcmtcnl cxpenrnents, wnh samples ttl each experiment run In triplicatc ·Values for trealed synaptosol11CS lilat arc sl)!nllicantly dillcrcnl IrOin control (P 0.05) ceramide treatment did not affect the Bnmx (250 ± 57.8, 227.3 ± 36 and 247.3 41 pmol"g for control, C2·cerarnide and C2-dihydroceramide, respectively) or Kt (9.3 ± 0.7, 16,7 and I 1.1 ± 1,5 nM tor control, C2-ceramidc and C2-dihydroceramide, respectively) of r3H]WIN3542R bind­ing. Results presented in !i~!,. indicate that the ceramidt'­induced decrease in r·1Hldoparnine uptake was reversihle since washmg of C2-ceramide-treated synaptosomes elimi­nated the decrease in r1H]dopamine uptake, In contrast to eOects on r1H Idopamine uptake, C2-ceram­ide application to striatal synaptosomes increased ['HIS-lIT uptake, The inactive ceraruide analog, C2-dihydroceramidc, had no significant eilect on 5-111' uptake at a concentration of 100 11M (values for striatal synaptosomal 5-tfT uptake were: control 147,2 15.3, C2-ceramide 229, I 6.4* and C2-dihydroceramide 135.2 10.7 finol/rug tissue; 1/=3; *P:s:; (>.05). * * * * F... /Ji 7-----T- r /~~..1j \\ \ • 1/ II , ~~~~- -tt- Vehicle i ____ Ceramide o 0.1 1 10 100 500 Dopamine (nM) 5_ Synaptosomcs were incuhated with C2-ceranlldc, DA amhlr vehicle lor 10 min pnor to prior to the addillOn of ['H15-HT. Values represent means and venlca! lines 1 S.E_M_ or detcnninations Irom three independent cxperimcnLs, with sample, in each experiment run in tnpllcatc. ~Valucs for C2-cemmidc-trealcd synaptosomc., Ihal arc sigmlicantly dtl1l:rcnt from concentration-matched controls ( P .; 0.05) 50 14 EL Riddl,. 1'/ al. ! El/ropean JOllrnal (~r Pharlllacologl' 458 (2()03) -' '-3(, Because the striatum contains hoth dopamine and 5-HT transporters, the next expcriments were designed to elucidate which transporter (dopamine or 5-1 IT) was responsihle lor the increase in synaptosomal 5-HT uptake. In a tirst experi­ment, 100 nM and 100 flM concentrations of citalopram were employed to selectively prevent 5-HT transport through the 5-IIT transporter (IC'.;o I.R nM) and/or dopamine trans­poner (IC~() 41 ~lM; Ilyth.:i. ! ')1-(2). Results presented in l-i~.' .. _, demonstrate that the cerami dc-induced increase in 5-11T uptake is blocked hy 100 11M citalopram (i.e., a concen­tralion sullicient to hlock hoth dopamine and 5-f1T trans­portcr function,. The ceramide-induced increase in 5-IIT uptake was not hloeked hy 100 nM cilalopram (i.e., a concentration sufficient to selectively inhihit 5-J-H trans­porter function). In contrast, application 0(" a relatively selective concentration (100 nM) of the dopamine reuptake inhibitor, methylphenidate (1('511 tor dopamine and 5-1-IT uptake of 165 and 260()() nM, respectively (Fleckenstein et ;;! _ i ()')O I), prevented the cerami de-induced increase in 5-11T uptake (11~f. ell. It is noteworthy that this concentration 0(" methylphenidate was not el1tirely selective, as it deereascd 5-HT uptake per se hy 22'% (fig. 4). To further address the issue of suhstrate specificity, the ability of dopamine to compete for ceramide-affected 5-HT uptake was examined. Results presented in h:!. 5 demon­strate that dopamine dose-dependently competes for this transport. Specifically, 500 n M dopamine attenuated the ceramide-induced increase in 5-HT uptake. Noteworthy, however, arc findings that this concentration decreased 5-HT uptake per se by approximately 33% (Fig 5). Results presented in Fii~. (l demonstrate that the ceramide­induced increase still occurs, even when applied to striatal synaptosomes prepared from rats treated previously with para-chloroamphetamine (7.5 mg/kg, i.p.), a dosing regimen * Saline [=--:-J Vehicle _ Ceramide peA peA \·lg. (I. Rats received porll-chloroamphelaminc (I'(,A; 7.5 ml;lkl;. S.l".) or salme vchide (I mt.'kg. s.c.) I week pnor to decapitation. Synaptosomes were prepared from the PCA- and saline-treated rub. and then incubated WIth 100 Ill"! C2-eeramidc or vehicle for 10 min prior to the addition of ['III~-HT Columns reprcsent means and veT1ical lines 1 S.E.M. of dcLennmatlons 10 live to six rats. ·Values lor peA-treated rats and/or ceramidc-trc;Jtcd synaptosomes thaI arc sll;niticantly different Irom control. /; Vallie for P( 'A- and cemmlde-lreatcd groups that arc signifIcantly from PCA-treated rats I P <,0.(5) that destroyed 5-IIT neurons (and presumably 5-HT trans­porters) as evidenced by a 57(~!() decrease in ['H J5-IIT uptake 7 days after drug treatment. In contrast to effects in striatal synaptoso'mes, C2-ceram­ide did not increase plasmalemmal [' H ]5-1I T uptake in hip­pocampal synaptosomes. Instead, ceramide tn.:atment de­creased ['1115-IIT uptake in synaptosomes prepared from bippocampal tissue (values tor hippocampal synaptosomal 5-11T uptake were: control 2.04 ± 0.10, C2-ceramide 1.23 ± 0,17 tino[iflg protein: IJ = 3). 4. l>iscussion Considerahle attention has been directed towards eluci­dating the mechanism underlying the regulation of dopamine and 5-HT transporters. In particular, it has heen demonstra­ted that phosphorylation decreases the activity, and leads to intemalization, of dopamine and 5-HT transporters (V;'lI\!iUll (:t a!.. 1')()7: ()ia!l cl a!., i9LJ7; Pri:;tup:: c\ <il._ I()()~'. Rarn:Hilllorthy and \shlkcly, 1 l)'-)l)) The data presented in this manuscript demonstrate that the lipid second messenger, cemmide, alters the function of dopamine transporters. The physiological importance of the cerami dc-induced changes in dopamine uptake is evidenced by findings that it is reversible. Specifically, application of C2-ceramide dec­reased striatal dopamine uptake and this phenomenon was reversed hy washing ceramide hom tht: preparation. In contrast to its effects on dopamine uptake, ceramide application increased 5-I--IT uptake into striatal synaptosomes (which contain hoth dopamine and 5-HT transpor1ers). The dopamine transporter transports dopamine readily, but has little affinity for 5-HT (i.e., the K, for 5-H1' inhibition of dopamine uptake is >10 11M: Giro:; ct al.. \ iN I). l310ckade or both dopamine and 5-HT transporters by 1 00 ~lM citalopram completely prevented the ceramide-induced increase in 5-11T uptake. However, blockade of the 5-111' transporter, but not the dopamine transporter, by 100 nM citalopram did not prevent the incrcased 5-HT uptake. Hence, these data suggest that the increased uptake of 5-HT by striatal synaptosol11es may be occurring through the dopamine transporter. Consistent with the hypothesis that the ceramide-indueed increase in 5-1--1'1' uptake is occurring through the dopamine transporter are tindings that the increase is prevented upon application of a relatively selective concentration (100 nM ) of the dopamine reuptake inhibitor, methylphenidate (the Ie,!) of methylphenidate for dopamine and 5-H1' uplake arc 165 and 260()O nM, respectively; tlcck-:nskill et :d .. i l)<)l)) These data are conii.Hmded, however, by the hlct that these concentrations of methylphenidate were not entirely selec­tive (c.g., application of 100 nM methylphenidate decreased 5-H1' uptake by 22%). Accordingly, these data suggest either some uptake of 5-HT by the dopamine transporter, or that 100 nM methylphenidate is preventing a small amounl of 5-1H uptake by the 5-HT transporter. Lower concentrations of methylphenidate (I nM) did not prevent the cerami de- 51 Riddle /'1 al. . European Journal of Phanll(lcolo!{1' 458 (2003) 3S induced increase in 5-I1T uptake (data not shown). Unlortu­nately, this concentration was without eHect on dopamine uptake per se and, therefore, render the 5-HT data incon­clusive. Since neither citaJopram nor methylphenidate are per­fectly selective as inhibitors of their respective transp011ers, and becausc reuptake inhibitors with absolute selectivity are not available, additional studies were conducted to test the hypothesis that ceramide is effecting 5-11T transport via th.: dopamine transporter. Accordingly, results presented in til'. ') dcmonstrate that ceramide still increases 5-IIT uptake, even after a substantial number of 5-HT transporters have been destroyed as a result of para-chloroamphetamine adminis­tration. These data arc confounded by the tact the pa/'{/­chloro< llllphelaminc lesIOn did not destroy all 5-HT projec­tions. Still, the finding that ceramide increased 5-HT uptake hy a similar magnitude in both saline-and para-chloroam­phctamme- treated rats is consistent with the hypothesis of altered substrate recognition by the dopamine transporter. Additional data presented in i support the hypothesis that cemmidc-induced increases in 5-HT uptake are mediared via the dopamine transporter in that dopamine per se com­peted ti.lr this uptake. Even more compelling is the finding that ceramide does not increase 5-HT uptake in hippocampal synaplosomes: these data are predictable since the hippo­campus is largely devoid of dopamine transporters and lhereliJre lacking targets upon which cemmide might act. Rather, ceramide treatment decreased 5-HT uptake. These data suggest thal ceramide may decrease 5-HT transport via 5-HT transporters, per se, or via other trnnsporters l<-lUnd in the hippocampus (i.e., the norepinephrine transporter). Since ceramides activate both protein kinases and phos­phatases, the ceramide-induced changes in dopamine uptake may be due to altered phosphorylation of the dopamine transporter. The dopamine transporter contains many con­sensus phosphorylation sites and, as noted above, activation of protein kinase C by phorbol esters decreases dopamine transporter activity and leads to intemali71ltion of the trans­porter. Accordingly, the ceramide-induced decrease in dop­amine uptake could be explained by phosphorylation and intemahzHtion of the dopamine transporter. This hypothcsis is not, however, sufficient to explain all 1)1' the data gince the ceramide-induced increase in 5-1·IT uptake would not occur if transporters were internalized. Moreover, the lack of effect of ceramide Oil WIN3542X binding is not consistent with internalization, assuming that W[N3542l:i is not membrane­penneable. It is possible that differing phosphorylation sites mediate differing eflects (i.e .. internalization vs. substrate recognition). In addition, the cemmide-induced effects on thc dopamine transporter may represent a novel phosphoryla­tion- independenl mechanism of transporter regulation. Fur­ther studies arc necessary to elucidate mechanisms whereby this lipid second messenger atlccts monoamine transporter limction. In summary. we conclude that cerami de decreases dop­amine uptake through the dopamine transporter. In addition, ceramide appears to increase 5-tn uptake through the dopamine transporter. Noteworthy arc recent studies dem­onstrating 5-11T transport into dopamine neurons (Suare;­Roca and Cub.:ddu. Zhou ::00:::.1. The present data extend these findings hy suggesting that such uptake may be enhanced hy ceramide treatment. These data are the tirst to demonstrate that transporter function can be rapidly and reversibly regulated by a lipid second messenger. Acknowledgements This study was supported by NIH grants Ui\ 14475. DAOOX()9, DA 113X(). DA0037f1 and DAO·l222. References £lli[!h. Dyer, W.J. 1959. A rap1(j mcthod of tolal lipId ~XlraclHln and purificallon. Can. 1. Biochcm. Phy~iol. 17. 911 917 Carvelli. L. Moron. lA .. Kahli~. K.M .. Ferrcr. lV.. Scn. N .. l.cchlcllcr. J.fL Lech-Lundberg. L.M. Merrill. (i..l.ater. I.:.M .. Ballou.I..M. Ship­pen berg. T.S., JavllCh. LIIl. R.I.. GallI. A .. 2002. PI 3-kinasc reg­ulation of dopammc uptake . .I. Ncumchcl11. X I. R~9 - XIl£) Copeland. IU .. Vogclshcf!!. Y., Neff. N.H .. Hadllconstantmou. M .. 19'16. Protein kinase (. activators decrease dopamine uptake imo stnatal SYII-J. Pharmacol. Exp. Ther 152" - 1532 R.T.. Ilannun. Y.A .. 1992. Ccral!llde stImulates a cystnhc protein phosphatase. J. Bioi. Chem 504x - SO'; I Fkckenstcin. A.E .. Metzger. R.R., Wilkins. D.G .. Gibh. J.W _ Hanson. O.R. 19'J7. Rapid and reversihle cftcet~ of methamphetamine on dop­. I PhamlJCol. Exp. TI,Cr. 2X2. 104,i\3X. 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R.A .. Hanson. (i.R .. Fleckenstein. lI.E.. l'!lJX Nature or methamphetamine-induced rapid and rcv~rsiblc "hangc;; m dorammc transporters J. l'harmacol. 361. 269, c7~ Oka;,:aki. T .. Bielawski!. A .. Beil, R.M .. Hannun. 1'.A .. J9'10. Role of ccralllitie 'lS a lipid mediator or la.2S-dihydroxyvitarnin D,-induced HI.,6(j cell tiillcrcmiatiol1 . .i. BioI. ('hem 265. 15X23-·15X~1. Preiss, J., LoomiS. CoR .. Bishop. w.R .. Stem, R .. Niedd. J.E .. Bell. R.M. 19X6. Quantativc measurement or sn-I.2-diucylglycerol~ prcsent In pla­IdcL~. hepatocyte;;, and ras- and ci,-transJllrllled normal mllminey cclJ\ .i. BioI. Chclll. 261, X5'17 .. X600 l'ristupa. Z.B .. McConkey. r.. Lill. Man. fLY. l.ee. F..I .. Wall!!. Y.T., Ni,mik. H.B., 19%. Protein kinase-medtated bidIrectional tramckin~ and limctional rc)!ulation of the human dopamine transporter. Synapse 30.79 - 1'.7. Qian. Y. Galli, A .. Rumamoorthy. S., ItlsSO. S .. DeFelice. I...J .. Hlakely. 52 EL Riddle cl al. ! t.IINlp<'all JOt/mal of Pharmacology 451:1 (:t003) 31 ·31S N IL 1997 Protein kinase C activation rc!:ulates human serotonin tr,msporicrs in IIEK-293 cells via ahen:d cell surface expression. J Ncurosci !7. RUlllDmoorthy. Blakely. R.D .. 1999. PhosphorylatiolJ lmd sequeslr"hon or serotonm transporter:; di Ili:rcntially modulated hy psychostllllulant<; SCience SietslllJ.IL Veldman. RJ .• Kok.1W.. 200!. The involvement ofsphinllO-lipids in resistance. 1 Mcmbr Bio!. IXl. 153 162 Suan:z·RlIea. If.. 200} The selective serotonin reu[ltakc illillhltor cHaln[lram mducc~ the storag..: or serotomn III eatcdwlalllincr­! L'" tertlllnuis. J Phnnnaco!' bp. Ther. 302. 174- 17'>. Tanahe. L. Cui. S .. Ito. M .. 199K. ('cramldc rrolllotes calpain-mcdtatcd I'rnh~nly-'is oi' protein kina~c (' I', 111 murtne polyfJlofj1honucicar Icukn­cyk" Bioch<.:ll1. 810pl1ys. Res. l'oIllIllUIl .. ~42. 12'l 133. Vaughan. R)\ .. Hull. R.A .. Uhl. G.R .. Kuhar. M . .L. 19'P, Protein /..ina.'" ( . -Illetlllltcd phosphorylation and functional regulation or dopamlilc lr<:l!l.SpOrlcrs 1T1 slnatal synaptosOIm:s. J. UIOL ('hem 272. 1."541-15541,. Wcslwick. 1.K .. Bi~law"ka. A.E .. Dbaibo. Ci .. Hannun. Y A. Hrcnncr. /).A .. 1995. Cernmidc activates the stress-activat.:d protClIl kinases J. BioI. Ch.:m. 270. 22689 .. 22692 Whatley. R.E. Stroud, ED .. Bunting. M .. ZmlnlCrtnan. (lA. Mcintyre. TM .. Prescot!, S.M .. 1993. Cirowth-dcrentlcn! changes trl araehid,Hlle add relc;"c from endothelial cells arc mediated hy rrntdn kinas," C and in dwcylglycerol. J Bioi ('hem j(, 130· 1(,1]X Zhnu. .. Lesch. KP .. Murrhy. 1).1.. 2002. Serotonin Ur1ake inlo dop-amine lIeurons via tloramine transrorters: a cOlllpcnsatory alternative. Brain Res. 942. lOt) .. 11 ') Zhll. Kavanaugh. M.P .. Sonde..,;. M.S .. Amara. (i. Zahmscr. N R .. J 997. Activation of protem kinase (. inhihits uplake. current, and bind­ing aSSOCiated with the numan dopamme lransrnrter c1(pr(~ss~d III xen-opus (locytes. J Phanllaclli. Lxr. Thel lJ5X 53 Foreword CHAPTERS DIFFERENTIAL TRAFFICKING OF THE VESICULAR MONOAMINE TRANSPORTER-2 BY METH· AMPHETAMINE AND COCAINE In dopaminergic neurons, synaptic transmission occurs as dopamine (DA)-filled synaptic vesicles undergo exocytosis at the presynaptic nerve terminal. In order for this to occur, the synaptic vesicle must have participated in multiple processes. Some of these processes include vesicle formation, filling of the vesicle with DA, and trafficking of the vesicle. Although many advances have been made in understanding the cycling of synaptic vesicles, this process remains poorly understood. Synaptic vesicles utilize many proteins which allow them to take up DA and move about the neuron. The vesicular monoamine transporter-2 (VMAT-2) is the sole protein responsible for the transport of cytosolic DA into the vesicle (for review, see Schuldiner, 1994). However, many proteins are required for the trafficking of vesicle cycling (Augustine et aI., 1999). Recently. our laboratory (Brown et aI., 2000; Brown et al 200 1) and others (Hogan et aI., 2000) demonstrated that psychostimulants rapidly alter the amount of DA uptake into synaptic vesicles prepared from rodent brain tissue. Interestingly, 55 amphetamine analogs decrease (Brown et aI., 2000; Appendix C), whereas cocaine and methylphenidate (MPD) increase (Brown et a1., 2001; Appendix D), the amount of DA taken up by these purified synaptic vesicles. These observed alterations in vesicular uptake could reflect a difference in the transport capacity of the vesicular monoamine transporter-2 (VMAT-2) or a change in the quantity of vesicles purified. As a first step toward assessing the latter (i.e., whether amphetamine analogs and cocaine alter vesicle locaJization), the effects of cocaine and methamphetamine on synaptic vesicle distribution were investigated. These results demonstrated that the VMAT-2 protein, and presumab1y synaptic vesicles, are redistributed differentially after psychostimulant treatment. The implications of these phenomena, as well as the differential redistribution of VMA T -2 induced by amphetamine and MPD (Chapter 6) are discussed in Chapter 7. Augustine GJ, Burns ME, DeBello WM, Hilfiker S, Morgan JR, Schwiezer FE, Tokumaru H, Umayahara K (1999) Proteins involved in synaptic vesicle trafficking. J Physiol 520:33-41. Brown JM, Hanson GR, Fleckenstein AE (2000) Methamphetamine rapidly decreases vesicular dopamine uptake. J Neurochem 74:2221-2223. Brown JM, Hanson GR, Fleckenstein AE (2000) Regulation of the vesicu1ar monoamine transporter-2: A novel mechanism for cocaine and other psychostimulants. J Pharmacol Exper Ther 296:762-767. Hogan KA, Staal RG, Sonsalla PK (2000) Analysis of VMAT2 binding after methamphetamine or MPTP treatment: disparity between homogenates and vesicle preparations. J Neurochem 74:2217-2220. Schuldiner S (1994) A molecular glimpse of vesicular monoamine transporters. J Neurochem 62:2067-2078. Reprinted with permission from the European Journal of Pharmacology. ELSEVIER European Journal nf Pharmacology 449 CW()2) 'J I 74 Ww\\ .dsevlcr.nlnl.:locale:clpila. Short communication Differential trafficking of the vesicular monoamine transporter-2 by methamphetamine and cocaine Evan L. Riddle'\ Matthew K. Topham b, John W. Haycock c, Glen R. Hanson ", Annette E. Fleckenstein a.* I'Julrmf.If'{)I,w,· tmd l()xi('()logl~ (/1I11'I'I'SI/1 o{ lfIak 3() SOIlII! ?{)(JO EaSI RII!. :01 Sail 1.<lA,' Cill'. I 'T 8-1 I I:;. I 'SA Call('('I'llIslilll/e. (;niver.lII\' ,,{Ulab. Sal! l.a!.." ('I/,l'. LT US.·! '/kparllr/elll 01 Biucin·JlII."')' <llId Molecular Biulng)'. LOllislt/llt1 SUlI!' (illiwl'sil\' I/""ilil SciC'lI .. ".I' ("1111'1: /1,'('1\ Or/,·(IiI.\. LA. ('.',./ R"ccm:d 7 March 200]: accepted 14 June 2002 Abstract High-dose administration of cocaine or methamphetamine ttl rdts acutely (::0 24 h) alters vesi<.:ular oopamine transpnrt. This study elucidates the nature of these changes. Results reveal a differential redistributlOl1 of the vesicular monoamine transporter-::! (VM Al~2) withtn striatal synaptic tenninals after drug treatment. In particular. cocaine shift:,; VMAT-2 protem from a synaptosomal membrane traction to a vesicle-enriched fraction. as assessed ex vivo m fractions prepared from treated rats. In contrast. methamphetamine treatment redistributes VMAT-2 trom a vesicle-enriched fraction to a location that not retained in a ;;ynaptosomai preparation. These data suggest thaI psyehostimulants acutely and diflcrentially ailed VMAT-2 subcellular localization. ~', 2002 Elsevier Science B. V. All righls reserved. hr\·H'OI-d.I" VMAT-2 (veSicular nlOuoanHl1C Iransporter-2): Cocaioe: Methamphetamine: Traflkking: (Rat) I. Introduction The vesicular monoamine transporter-2 (VMAT-2) is the sole transporter responsible lor sequestration of intraneuro­nal monoamines. Amphetamines, presumably including methamphetamine. profoundly affect dopamine storage in synaptic vesicles (Sulzer el :11.. 1<)95; ell bells et al.. \99/.l-J. It has been suggested that intraneuronal sequestration of dopamine protects against autooxidation of excess intra­neuronal dopamine, which in turn may damage intracellular structures and compromise function (La Voic and llastings, 14')lJ; Fleckl:nslcin el al. 2()(1{)). High-dose methamphetamine administration decreases VMAT-2 ligand binding in rodents, as assessed in whole tissue preparations days after drug administration (I logan ct uL. 2000: Frey l't :d.. 14(7). These long-tenn reductions haw been used as an index of neuronal cell loss. Until recently, psychosllmulant-induced changes in VMAT-2 pro­tein levels have not been reported at early time points (i.e. -::;24 h) aner methamphetamine treatment. However. recent " CorrespondIng author Tel. + l-x()I-5li~-7474: lax: + I·SOJ-SHS-Slll. E-f1/mj "ddrl·.","' lleckCllstem'II'hsc.utahccdu IA.E. Fleckenstein). work employing subcellular fractionation has demonstrated changes in both VMAT-2 activity (assessed by measuring vesicular dopamine uptake) and dihydrotetrabenazine (a VMAT-2 ligand) binding in purified synaptic vesicles I h and J day al1er drug treatment (Brmm cilJl., 200(); Ih.lgan et at.. 200t),. For instance, our laboratory has demonstrated that in vivo administration of methamphetamine. as well as another psychostimulant, cocaine, decreases and increases dihydrotetrabenazine binding, respectively, in a purified vesicullJr preparation (Brown et al.. 200 I a,Il). Although the mechanism(s) by which cocaine and meth­amphetamine influence VMAT-2 is(are) not known, stimu­lant- induced redistribution of the VMAT-2-containing vesicles may provide an explanation. Hence. the purpose of the present study was to detemline if the change in dihydrotetrabenazine binding caused by methamphetamine and cocaine treatment is due to changes in the subcellular distribution of the VMAT-2. The results suggest thai cocaine and methamphetamine ditrerentially affect VMAT-2 subcel­lular location and, presumably. synaptic vesicle distribution. Specitically, cocaine treatment redistributes VMAT·2 from a synaptosomal membrane to a vesicle-enriched fraclion. In contrast, methamphetamine redistributes VMAT·2 from the OOI4-2999!02!S . sec Iront mailer 20()2 Ej,;cvicr Science ltV All rights rcscrwd I'll. SOOI4-29'!'!I(2)OI'lX)-4 56 72 EL Riddle el af. / EliroprYlll ./ollI"lJaI oj" Pharmacology 449 (2002) 7] - 74 P2 P3 53 Z-~:::hl .~ '§ 800 ~ ~ 600 't>~ ~:E 400 m ~2Q() o _'20 lUi ~~100 ~ § 80 * ~ ~ 60 't>~ C;!:: 40 . "'.0 m ~ 20 o '.~?':§~1 1,a25G00 l. uTt ~~90 't>~ c:~ 60 "'.0 m ~ 30 o Saline Cocaine Saline Cocaine Saline Cocaine Fig. 1 Cocaine alters VMAT-2 immunoreactivity in ~:lJhccliular fl-actions. Ral<; receiv~d a single administrntion of cocaine (30 mg/kg. i.p.) or ,'-:alinc vehicle (1 mL'kg. i.p .). All animal.s were sacrifIced III ai1cr the l~ocainc or saline injection. Columns represent the mean optic density. and error bars represent the S.E.M of delerminations in six treated rals. *Valuc.-; for cocaine-treated rats thot an; signi!icantly different from saline-treated controls (P s 0.05). vesicle-enriched fraction without significantly altering syn­aptosomal membmne VMAT-2 protein levels. These ditTer­ential patterns induced by cocaine and methamphetamine may underlie some of the different neurochemical and neurotoxic effects of these psychostimulants. 2. Materials and methods All experiments were conducted in accordance with the NIH Guidelines for the Care and Use of Laboratory Ani­mals. Where indicated, male Sprague- Dawley rats (weigh­ing 280 - 330 g) received a single injection of cocaine (30 mg/kg i.p.), multiple high-dose injections of methamphet­amine (4 x 10 mg/kg per injection, s.c., 2-h intervals), or saline vehicle (I mllkg per injection). Striatal synaptosomes were prepared tTom rats decapi­tated 1 h after treatment as previously described (Flecken­stein ct al.. 1997). Briefly, striatal tissue was homogenized in cold 0.32 M sucrose and centrifuged (800xg for 12 min; 4 'C). The supernatant (SI) was then centrifuged (22,OOOxg for 15 min; 4 'c) and the resulting pellet (P2, synaptosomal P2 fraction) was resuspended at 50 mg original wet weight/ml in cold water and a portion saved for Western blot analysis. The remainder of the synaptosomal sample was centrifuged for 20 min at 22,OOOxg (4 'c) to pellet lysed synaptosomal membmnes (P3, synaptosomal membrane fraction), which were then resuspended at 50 mg original wet weightlml and saved for Western blot analysis. Prior to resuspension of the plasmalemma I membrane fraction, the supernatant (S3, vesicle-enriched fraction) was removed and saved for West­ern blot analysis. Binding ofVMAT-2 antibody was performed using 60 fll aliquots of synaptosomal (P2), synaptosomal membrane (P3), or vesicle-enriched (S3) preparations. Each aliquot was added to 20 fll of loading buffer (final concentration: 2.25% sodium dodecyl sulfate, 18% glycerol, 180 mM Tris base (pH 6.8), 10% [,-mercapto-ethanol and bromophenol blue), boiled for 10 min, and loaded on a 10% sodium dodecyl sulfate-polyacrylamide gel. Following electropho­resis, samples were transferred to polyvinylidene difluoride membrane, blocked with 5% nonfat dry milk in Tris­buffered saline with Tween (250 mM NaCl, 50 mM Tris pH 7.4 and 0.05% Tween 20), and probed with the VMAT-2 P3 53 ~::::n. ~ 800 * C 600 'tI .C. 400 D:l 200 o 1000lDl ~ 800 c: ~ 600 -g 400 C'D" 200 o Z-:::[L ~ 1200 C 900 'tI c: 600 .. * al 300 o Saline METH Saline METH Saline METH I ••• •• ..-- Fig. 2. Methamphetamine alters VMAT-2 immunoreactivity in suhcellular fractions_ Rats received mulLiplc high-dose injections of methamphetamine (4 ): 10 mg/kg per injection, s.c., 2-h intervals). or saline vehicle (1 ml/kg per injection), All animals were sacrificed 1 h afkr the finnl methamphetamine or saline injection. C'olwnns represent the mean optic density. and error bars represent the S.E.M. of determinations in six trl.!atcd rat". *Valucs for methamphctaminc­treated rate; that arc significantly different from saline-treated controls (P ::; O.OS) 57 f,'L Riddl" ('/ al. European Journal of PlwrmaC'%gl' 449 (:!(J!)!j 74 antibody (provided by J.WI-L). Bound antibody was visual­ized with HRP-conjugated goat anli-rabbit antibody, and antigen antibody complexes were visualized by chemilu­minescence. Multiple exposures of blots were obtained to ensure development within the linear range or the film. Bands on blots were quantified by densitometry using Kodak 1 [) image-analysis software, 3. Results Results presented in Fig, ! demonstrate that a single mjection of cocaine nO mg/kg; i.p,) increases VMAT-2 imlllunoreactivity by 110% in the S3 (vesicle-enriched) frac­tion prepared from the striata of rats sacrificed I h after treatment. This increase was concurrent with a 33% decrease in the associated P3 (synaptosomal membrane) fraction. with no diJlcrt!nce he tween P2 (synaptosomal) fractions. Data presented in hg 2 demonstrate that I h after multiple high-dose administration of methamphetamine (4x 10 mgt kg; s,c.). VMAT-2 immunoreactivity in the S3 fraction was decreased by 80% compared to saline-treated controls. This decrease was concurrent with a 400;(, decrease in the 1>2 fraction and no ditlerence in the P3 ftactions. 4. Discussion Cocaine (Brown et al.. 200Ia.h) and methamphetamine (BruWIl <:1 al.. 2000; lIo1!:.m d 'II.. 20(0) increase and decrease, respectively. dihydrotetrabenazine binding and dopamine uptake as assessed in a purified vesicular prepa­ration. Explanations that may underlie the psychostimulant­induced changes include: (I) conronnational changes that alter VMAT .. 2 lunction and dihydrotetrabenazine binding; (~) changes in the turnover (degradation or synthesis) of the VMA.'J'-2: and (3) redistribution (trafficking) of VMAT-2 andtor VMAT-2-containing synaptic vesicles. The possihil­ity of confonnational changes can be largely eliminated as antibody binding and Westem blot analysis. using a dena­turing gel. are not influenced by the confonnational stale or the VMAT-2. or by the internal vs. external expression of the protein (i.e. lipid membranes are dissociated and pro­teills arc denatured). The possibility that VMAT-2 synthesis or degradation contributes to the present results also seems unlikely given the rapid nature ofthe effects (i.e. at 1 h aftcr cocaine or methamphetamine treatment). This is supported by findings that the II f(x monoamine transporter and receptor tumover is greater than I day (i.e. the tin iiJr dopamine receptor and transporter are 2- 3 and 6 days. respectively; Nonnilll et al.. 1987: Ballaglia el aL. ! 9XX: I·it:d:enstein et al.. 1(96) and by lindings in human and animal studie~ that there are no significant changes in total VMA.'r-2 protein levels measured 1 day atter cocaine or methamphetamine exposure (£logan et al.. 2(1)0: Wilson \.,t al.. !'J96a.bl. Instead of alterations in cOl1tl)nnalion. synthesis, or degradation, results presented ill this study suggest Ihat redistributIOn of the VMAT-2 protein, and presumably synaptic vesicles. is the mechanism likely responsihle lor the changes in the VMAT-2 in the purified vesicular preparations atter cocaine or methamphetamine treatment. In particular, cocaine administration causes a redistrihution or VMAT-2 protein from the P3 10 the S3 fraction. III these experimenls, the total amounl of VMA.'I~2 protein in the P3 in untreated animals is "" 70% of that found in the P2 fraction (dat(l not showl1). IIence. the relallvely small decrease in P3 VMAf"-2 immunoreactivity aller cocaine treatment would be expected to result in a large Inaeasc in S3 (!riven that the total amount of protein in the "2 fraction is not altered by cocaine treatment). Previous studies demonstrated that tOlal VMAT2 le"els arc not changed hy cocainc administration in brain homogenate or slice preparations. Our data arc consistent with these pre­vious data since no changes in total synaptosomal VMAT-2 were detected aller cocaine treatment. Moreover, our data demonstrate that cocaine may redistribute VMAT·2: a phe­nomenon that would not be detected when assessing total VMA1~2 protein levels. In contrast to the effects of cocaine on VMAT-2. results presented in Fig. 2 demonstrate that methamphetamine treatment largely decreased VMA.'f-2 immunoreactivity in the S3 fraction, This was concurrent with a moderate decrease in P2 VMAT-2 and no change in (>3 VMAT-2 levels. This decrease in S3 and P2 VMAT-2 may suggest tratlicking Irom the P2 traction altogether (i.e. traflieking out of the portion of nerve tenninal retained in a synapto­somal preparation) since decreases observed in the P2 and S3 Iraction arc not likely due to degradation of protein (Hogan et a!.. 2000; Wilson el al.. IY\)6a). Interestingly, amphetamine increases the phosphorylatiOTl or synapsin, thercby dissociating vesicles trom actin filaments (Iwata t:l al., 1996. ! YY7), Competitive inhibition of synapsin (a phenomenon that would presumably mimic synapsin phos­phorylation) reduces the number of synaptic vesicles within the nerve tenninal (Augus1int: el ai., 19(9). The location to where the VMA.'f"-2 is distributed remains to be investigated In summary, results demonstrate that cocaine and meth­amphetamine difterentially altcct thl: subcellular distribution of VMAJ"". .. 2, and presumahly synaptic vesicles. These drugs differentially affect the tranieking of VMAT-2 with respecl to the S3 fraction (methamphetamine out of and cocaine into). which suggests that these drugs diflcrentially alter trat1icking of vesicles between diJlerent cellular locations. It has been previously suggested that whole tissue VMAT-2 protein levels are a good indicator of neuron integrity; however. this does not imply that VMAT .. 2 is static amI lacks intracellular regulation. In fact. the present data demonstrate that VMAr-2 can be differentially redistributed among subcellular fractions. These acute changes in VMAT- 2 protein (that are not apparent in whole tissue preparations) may contribute to the differential behavioral profiles, neuro- 58 F,.I. Riddle t't al ; Europeall JOllrnal (~f PllllrmacoilJKI' "-If) (2002i 7!, 7./ toxicity, and abuse patterns induced by methamphetamine and cocaine. Acknowledgements This work was supported by NIl! grants DA 14475, DAO()X69, DA04222, and DA 113l<9. Refercn('cs '\U;:IISline. G.J.. Burns. M.L. I.kBello WM .. Hilliker, S., Morgan. J.R., Schweizer. F.E. TokullIaru.lL. lImayalJaI<!. 1\..1')99. PfIIlcin; involved III svnaptlc v",;id" trall'lckill)!. J. I'hysinl. 520. D·· 41 llallagllll, \I., Norman. A.I!.. Cree,;.:. I.. 191(1'. A!(c-rclated ditl~rcnlial re· covery rate,; or rat striatal D· J dopamine reccpton; lilliowing irreversi­ble lJ1actlvatlOl\. f:<lr. J. Pharmac,,!. 145. 2Hl -290 Brown . .I.M. Hanson. (;.R .. Fleckenstein. AE. 2000. Mcthamphela­mille rapIdly decreases vesicular dopamme uptake. 1. Neurochem. 74. :!221- 2223 Brown. 1.M., Hanson, U.K. Flcckcnstelll. AL. 1110 I a. ('ocaine-induced lIlercases III vesicular dopamine uptai--e: role of dopmllin~ receptors. J. PharmacoL Exp. Thcr. 2'1X. 115(J- 1153. Brov-1], J.M .. Hanson, G.R .. Fleckenstein, A.E.. 2001 h. Rel;!ulation of the vesicular monoamine transporter':!: a novel mcchallIsm li)r cocaine and other psycJlO.,tlnmlants. J. Pharmacnl. Exp. Tiler 196. 762· .. 767 Cubeils. J.F. Raypori. S .. Rajendran. Ci .. Sulzer. D .. 1(,194. Methamphet­amine neurotoxicity involves vacuolation or endocytic organdies and dopamilH:-dcpcndent intracellular oxidative stress. l Ncurosci. 14. 2260 - 2271 Fkckcllstcin. A.L. Pogun. S .. Carroll. F.L. Kulmr, M.L 1 ()Q6. Recovery of dopamine transportcr hillding anti function alter IIltra.~triatal administra· lion of the irreversible inhibitor RTI,76 :3iH3-p-chlnmphcnyl)tropan- 21\ ('arbnxylic aeid p-Isnlhiocyanatophenylethyl ester hydrochlondc; J Phannacol. [:xp. Ther. 279. lOU - 206 Fleckenstein. A E. Metzger, R.R .. Wilkins. D.(i .. Uibn. 1.W .. lIanson. (l.R .. 1997. Rapid ilnd rcversibk clrects or mcthalllphetamm~ on dop­amtn~ trmL"pOriCfS. 1. PharmacoL Exp. TIler. 21\2. X34 .. lOX Fleckenstein. A.£O .. (iibb . .I.W .. Hanson. G.R .. 2()()O. Di!Terential clrcets or stimulants om mOlloamincr;:lc transport~r.;: pharrnacologu;al consequen­ces and Implications I(lr neurotoxicity EUL l PharmacoL 406. I·· I, Frey. K. Kilhoum. M. Robinson. 1'.. 1997. Rcdu~ed strialal veSIcular monoamine uncI. neurotoxic bUI nol aller hchablOrally-scn-sitizing do!;es Eur. J I'hanllrlco/. 13·1 '}7:l Hogan. KA .. Staal. R.ll.W._ Sonsalla. PK .. 2U()(). :\nalysis ~I: \'MAr2 billding alter or MPTP trcntrm:nl: dIsparity hdw~cn hmnogcnates and prcparawms ~curochcm. 7·1, n 1 7 2220 Iwala, Hewlett. (aL Ferrell ST. Czcrnik. A.J.. Mein. K.F. (incgy. ME .. 1996 Increased in vilo phosphorylation state 01 m:llflllllodulin 110 sirialllm limn rats treated with repealed al1lphelamilw Exp. Thcr 142,,· 1434 Iwata. S .. Hewlett. G.l·L Uncgy. M.E .. 1')97. Amphdanllnc increases the phosphorylatiOn of neuTolllodulin and synar~in I [11 rat strialal SYlluptn­somes. Synapse ;Y" 2X I 291 LaVoie. M.1 .. Ha.~tm!:!s. T.(I .. 19'1'1. I'cmxynitritc- and nitnte-induccd OXI­dation of dOpallllne: implicallons ()r mtric OXIde in dopamincrgll' cell loss. J Ncurochelll. 73, 2546· 2'~4 Nonnan. A.B .. Battaglia. G .. Creese. L. 1!JX7. Differential recollerv rates of rat D2 dopamine receplors as a tuncliOIl or a;:ing and chronic 'reserpine treatment !ollowin;: irreversible modification: n key to receptor rc/!uia-tory mcchani~ms. J Ncurosei 14H4- 1491 Sulzer. D .. Chen. TK .. Lau. Y.Y.. Kristensen. H .. Rayp0ri. S .. 1'l'J5. Amphetamine redistributes dnpamint Irom synantlC the cytosol and pmmoi(.'s reverse tmnspon. 1 Ncun),~ci.· 4102 410X Wilson. 1.M .• Kalasinsky. K.S .. Levey. A.L Bergeron. C. Reiber. (i .. An­thony. R.M .. Schmunk. G.A .. Shannak. K .. Haywck. 1.W .. Kish. SJ .. l'i%a. Stnatal dopamine ncrve lenninal markers in human. chromc mClhamphetmninc users. Nat. Mcd. 2. 6<)<), 703 Wilson. J,M .. Levey. A.1.. Bcr!!eron, t' Kalasinsky. K .. Ang. L. Peretti. r , Adams. VI.. Smialck. L Anderson. W.R .. Shannak. K .. Deck. L NiL­nik. HR. Kish. S.J .. IQ96b. Striatal dopamine. dopal11mc transporter, and vesicular monoamine transporter in chronic cocaine users Ann NeuroL 411. 42X-439 59 Introduction CHAPTER 6 SELECTIVE TRAFFICKING OF THE VESICULAR MONOAMINE TRANSPORTER .. 2 CAUSED BY AMPHETAMINE AND METHYLPHENIDATE As reported in the previous chapter, a single high-dose administration of cocaine and multiple high-dose administrations of methamphetamine differentially alter vesicular monoamine transporter-2 (VMAT-2) distribution. In an additional manuscript (Appendix D), it was reported that a single high-dose (40 mg/kg, s.c.) administration of methylphenidate (MPD) traffics vesicles in a manner somewhat similar to cocaine. In particular, cocaine and MPD decrease the amount of VMAT-2 in the P3 (synaptosomal membrane) fraction while increasing it in the S3 (nonmembrane associated) fraction. Neither the specificity of these phenomena nor mechanisms underlying them have been determined. Synaptic vesicles are organized into two distinct functional pools: a large reserve pool in which vesicles are restrained by the actin-based cytoskeleton, and a quantitatively smaller releasable pool in which vesicles approach the presynaptic membrane and eventually fuse with it upon stimulation (Benfenati et aI., 1999). Synapsins are synaptic vesicle-associated proteins that are involved in maintaining synaptic vesicles in the 61 reserve pool (Humeau et aI., 200]; Hilfiker et aI., ) 999), a process regulated by (de)phosphorylation of synapsin (Benfenati et aI., 1992). Cocaine and amphetan1ine differentially alter the phosphorylation state of synapsin (Iwata et aI., ] 996; Smith et aI., 1993), allowing for the speculation that psychostimulants-induced vesicle trafficking is mediated by synapsin. The focus of the studies presented in thjs chapter is to determine: 1) if low, clinically relevant doses, of amphetamine (AMPH) and MPD alter VMAT-2 trafficking; 2) if the psychostimulant-induced trafficking occurs with other, non-VMAT-2 containing synaptic vesicles in the striatum: and 3) if synapsin is involved in this trafficking process. Results demonstrate that no changes were detected in the phosphorylation of serine 9 of the vesicle trafficking protein synapsin 1. However, clinically relevant doses of both AMPH (Richards et aI., 1999) and MPD (Kuczenski and Segal, 2002) differentially alter VMAT-2 trafficking. This effect is specific for VMAT-2 in that vesicular glutamate, acetylcholine, and GABA transporters were not altered by high- or low-dose administrations of AMPH or MPD. Materials and Methods Animals. All experiments were conducted in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals. Where indicated, male Sprague­Dawley rats received a single injection of amphetamine (2 or 15 mg/kg. s.c.), methylphenidate (2 or 40 mg/kg, s.c.), or saline vehicle' (1 ml/kg). Drugs and chemicals. (±)MPD hydrochloride and d-amphetamine were supplied by the National Institute on Drug Abuse (Bethesda, MD). Okadaic acid was purchased 62 from Calbiochem (La Jolla, CA). The VMAT -2 and phosphosynapsin antibodies were purchased from Chemicon (Temecula, CA) and Cell Signaling Technology (Beverly, MA), respectively. Vesicular glutamate, acetylcholine, and GABA transporter antibodies were generously provided by Robert Edwards (University of California San Francisco, CA). Preparation of subcellular fractions. Striatal synaptosomes were prepared from rats decapitated J h after treatment. Striatal tissue was homogenized in cold 0.32 M sucrose and centrifuged (1000 x g for 10 min; 4°C). The supernatant (S I) was then centrifuged (l0,000 x g for IS min; 4°C) and the resulting pellet (P2, synaptosomal fraction) was resuspended at 50 mg original wet weight/n11 in cold water and a portion saved for western blot analysis. The remainder of the synaptosomal sample was centrifuged for 20 min at 25,000 x g (4°C) to pellet lysed synaptosomal membranes (P3, synaptosomal membrane fraction), which were then resuspended at 50 mg original wet weight/ml and saved for western blot analysis. Prior to resuspension of the synaptosomal membrane fraction (P3) the supernatant (S3) was removed and saved for western blot analysis. Western blot analysis. Binding of VMAT-2 antibody was performed using aliquots containing 50 ~g protein of synaptosomal (P2), 30 ~g protein synaptosomal membrane (P3), or 20 ~g protein vesicle-enriched (S3) preparations. Each aliquot was added to loading buffer (final concentration: 2.250/0 sodium dodecyl sulfate, 18% glycerol, 180 mM Tris base (pH 6.8), 100/0 0-mercapto-ethanol and bromophenol blue), boiled for 10 min, and loaded on a ] 0% sodium dodecyl sulfate-polyacrylamide gel. FolJowing electrophoresis, samples were transferred to polyvinly I idene difluoride 63 membrane, blocked with 5% nonfat dry milk in Tris-buffered saline with tween (TBST~ 250 mM NaCI, 50 mM Tris pH 7.4 and 0.05°10 Tween 20), and probed with the VMAT-2 antibody. For binding of the phosphosynapsin antibody polyvinylidine diflouride membrane was blocked in 5% BSA in TBST, fo1Jowed by probing with the phosphosynapsin antibody in 50/0 BSA in TBST. Bound antibody was visualized with HRP-conjugated goat anti-rabbit antibody, and antigen-antibody complexes were visualized by chemiluminescence. Multiple exposures of blots were obtained to ensure development within the linear range of the film. Bands on blots were quantified by densitometry using Kodak 10 image-analysis software. All protein concentrations were determined by a Bio-Rad (Hercules, CA) protein assay. Results Figure 6.1 demonstrates that a single high-dose injection of MPD (40 mg/kg, s.c.) reduces the amount of VMAT-2 in the P3 fraction while increasing it in the S3 fraction. In contrast, a single high-dose administration of AMPH (15 mg/kg, s.c.) decreases the amount of VMAT-2 in the S3 fraction (Figure 6.2). Figures 6.3 and 6.4 demonstrate that similar trends are seen after administration of clinically relevant (Richards et aI., 1999~ Kuczenski and Segal, 2002) doses of MPD (2 mg/kg, s.c.) and AMPH (2 mg/kg, s.c.). The effects of the 2 mg/kg dose of MPD appear maximal at 1 h and return to normal by 2 h (Figure 6.5). Similarly, the effects of a 2 mg/kg dose of AMPH appear maximal at 1 h, but return to normal by 4 h (Figure 6.6), The vesicular protein redi stributions produced by low or high doses of MPD or AMPH are specific for VMA T -2 in that neither cholinergic, GABAergic, or 64 Figure 6.1. A single high-dose administration of MPD altered VMAT-2 subcellular localization. Rats received a single administration of MPD (40 mg/kg, s.c.) or saline vehicle (1 ml/kg, s.c.). All animals were sacrificed 1 h after the MPD or saline injection. Columns represent the mean optic density, and error bars represent the SEM of determinations in six treated rats. *Values for MPD treated rats that are significantly different from control (p ~ 0.(5). 65 0 c.. M ~ en CI) r:::: co en 0 0 0 0 0 0 0 0 0 0 0 I.{) 00 or- "<:t ,....... ('I) C\J C\J 0r-o c.. M ~ a.. CI) .-rc:-:o:: en 0 0 0 0 0 0 0 0 0 0 0 I.{) C\J Q') <D ('I) or- 0r- o c.. N ~ C. CI) r:::: co en 0 0 0 0 0 0 0 0 0 0 0 I.{) 0 I.{) 0 I.{) C\J C\J T""" or- (SllUn AJBJllqJB) AlISUaa pUBS 66 Figure 6.2. A single high-dose administration of AM PH altered VMAT-2 subcellular localization. Rats received a single administration of AMPH (15 mg/kg, s.c.) or saline vehicle (1 mllkg, s.c.). All animals were sacrificed I h after the AMPH or saline injection. Columns represent the mean optic density, and error bars represent the SEM of determinations in six treated rats. *Values for AMPH treated rats that are significantly different from control (p ~ 0.05). 67 :I: D.. :E M <C en Cl) .-ecan-::s 0 0 0 0 0 0 0 0 0 0 0 LO 0 LO 0 LO (\J (\J or- T""" :I: D.. :E M <C D.. Cl) .-ca-::s en 0 0 0 0 0 0 0 LO 0 LO or- T""" :I: D.. :E N <C D.. Cl) .e-can-::s , 0 0 0 0 0 0 0 0 0 0 LO 0 LO (\J T""" ,..... (sllUn ~JeJllqJe) ~IISUaa pues 68 Figure 6.3. A single low-dose administration of MPD altered VMAT-2 subcellular localization. Rats received a single administration of MPD (2 mg/kg, s.c.) or saline vehicle (1 ml/kg, s.c.). All animals were sacrificed 1 h after the MPD or saline injection. Columns represent the mean optic density, and error bars represent the SEM of determinations in six treated rats. *Values for MPD treated rats that are significantly different from control (p s 0.05). 69 0 1. D.. , M :: en Q) .-c-: CO en 0 0 0 0 0 0 0 0 0 0 0 0 0 0 LO 0 LO 0 LO C") C\J C\J ,..... ,..... 0 D.. M :: a.. Q) .-cc-o: en 0 0 0 0 0 0 0 0 0 ,C..\.J.. 0') to C") 0 D.. N :: a.. Q) ~ c: ~ .e-cn-o t 0 0 0 0 0 0 0 0 0 0 0 LO 0 LO 0 LO C\J C\J ,..... ,..... (Sllun AJBJllqJB) Allsuaa pUBS 70 Figure 6.4. A single low-dose administration of AMPH altered VMAT-2 subcellular localization. Rats received a single administration of AMPH (2 mg/kg, s.c.) or saline vehicle (1 mIl kg , s.c.). All animals were sacrificed 1 h after the MPD or saline injection. Columns represent the mean optic density, and error bars represent the SEM of determinations in six treated rats. *Values for AMPH treated rats that are significantly different from control (p ~ 0.05). 71 ~ Q. ~ M <C C/) Cl) .-s::-:::: as en 0 0 0 0 0 0 0 0 0 0 0 LD 0 LD 0 LD C\J C\J ,- ,- ~ Q. ~ M <C c.. Cl) s:::::: -as en 0 0 0 0 0 0 0 0 0 0 0 LD <0 t-- CX) 0'> '<:;t C"') C\J ,- ~ Q. ~ N <C c.. Cl) s:::::: as en 0 0 0 0 0 0 0 0 0 0 0 LD CX) ,- '<:;t t-- C"') C\J C\J ,- (SIIUn AJBJllqJB) AIISuaa pUBS 72 Figure 6.5. A single low-dose administration of MPD rapidly and reversibly increased VMAT-2 immunoreacivity in the S3 (cytoplasmic) fraction. Rats received a single administration of MPD (2 mg/kg, s.c.) or saline vehicle (1 ml/kg, s.c.). Animals were sacrificed 30 min to 6 h later. ~ t.n. o 73 74 Figure 6.6. A single low-dose administration of AMPH rapidly and reversibly decreased VMAT-2 immunoreacivity in the S3 (cytoplasmic) fraction. Rats received a single administration of MPD (2 mg/kg, s.c.) or saline vehicle (1 ml/kg, s.c.). Animals were sacrificed 30 min to 6 h later. ...c tn . o 75 76 glutamatergic vesicular transporter proteins are redistributed (Table 6.1). The trafficking events associated with high dose administration of ANIPH or MPD did not alter synapsin 1 serine 9 phosphorylation (control 226.5 ± 24.3. 15 mg/kg AMPH 252.1 ± 26.6~ control 156.0 ± 15.3.40 mg/kg MPD 173.5 ± 17.7). Figure 6.7 demonstrates that incubation of striatal synaptosomes with 10 jiM okadaic acid for 30 min increased the phosphorylation of serine 9 of synapsin 1, but that no trafficking of VMAT -2 occurred after okadaic acid treatment. Discussion The trafficking of synaptic vesicles is a complicated process that involves many proteins (Augustine et aI., 1999). Although many of the mechanisms underlying this phenomenon remain unknown, psycho stimulants were observed to influence vesicle trafficking (Chapter 5; Appendices D and E). These reports demonstrate that psycho stimulants differentially alter the trafficking of VMA T -2-containing vesicles. Results presented in this chapter support the previous findings as well as demonstrate that these trafficking events are specific for VMAT -2-containing vesicles. This specificity is likely due to the fact that DA receptors are involved in these trafficking events (Appendix D). One protein that may be involved in the psychostimulant-induced trafficking of synaptic vesicles is synapsin. Synapsin is a protein that maintains synaptic vesicles in the reserve pool (Humeau et aI., 200 I; Hilfiker et aI.. 1999). The dephosphorylated form of synapsin tethers synaptic vesicles to F-actin, but upon phosphorylation the tethering is abolished and the vesicles may then be translocated to the release sites (Bahler and 77 Table 6.1. A single low- or high-dose administration of AMPH or MPD did not alter redistribution of vesicular glutamate (VGLUT-l ,2), acetylcholine (V AChT), or GABA (VGAT) transporters in the P2 (synaptosomal) or S3 (cytoplasmic) fraction. Rats received a single administration of AMPH (2 or 15 mg/kg, s.c.), MPD (2 or 40 mg/kg, s.c.), or saline vehicle (1 ml/kg, s.c.). Animals were sacrificed I h later. VGLUT-1 VGLUT-2 YAChT VGAT VGLUT-1 VGLUT-2 YAChT VGAT 2 mglkg ~IPD P2 89.7 + 6,4 92.3 + 4.4 90.5 + 5.1 100.6 + 15.5 S3 108.7 + 11.0 82.5 + 14.4 104.7 + 11.9 112.7 + 9.6 2mg/kgAMPH P2 130.3±10.4 116.6 + 7.7 107.3 4.8 100.1 + 6.2 S3 87.9 + 3.8 97.5 9.4 93.1 ±8.6 102.4 9.6 40 mgikgMPD P2 104.8 ± 6.1 95.6 ± 11.9 99.8 6.5 116.8 S3 104.9 + 11.8 113.8 ± 10.0 106.0 12.4 106.6 7.0 15 mg/kg AMPH 1