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MDMA (Ecstasy) Metabolites and Neurotoxicity On
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MDMA and Neurotoxicity
Kantor,
L., Gnegy, M. E.
Protein Kinase C Inhibitors Block Amphetamine-Mediated
Dopamine Release in Rat Striatal Slices
J Pharmacol Exp
Ther 1998 284: 592-598
"The stimulant drug amphetamine is postulated to
enhance dopamine release through the plasmalemmal dopamine transporter by an exchange
diffusion with synaptosomal dopamine. Because protein kinase C has been shown
to have an effect on dopamine transporter activity, we examined the effect of
protein kinase C inhibitors on endogenous dopamine release stimulated by amphetamine
in perfused rat striatal slices. At concentrations of 1 microM, the selective
protein kinase C inhibitors chelerythrine, Ro31-8220 and calphostin C nearly completely
inhibited endogenous dopamine release elicited by 1 microM amphetamine. The inactive
analog bisindoylmaleimide V had no effect. Extracellular Ca++ was not required
for the effect of the inhibitors. The importance of vesicular dopamine release
was examined by determining inhibitor activity in reserpine-treated rats. Dopamine
release elicited by 1 microM amphetamine was not significantly altered in reserpine-treated
rats compared with control animals. Ro31-8220 at 1 microM completely blocked amphetamine-induced
dopamine release in reserpine-treated rats. Activation of protein kinase C with
250 nM of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate increased dopamine
release, and the release was not additive with 1 microM amphetamine. Both chelerythrine
and Ro31-8220 at 1 microM increased [3H]dopamine uptake by 17% and 30%, respectively,
whereas a brief exposure to 12-O-tetradecanoylphorbol 13-acetate slightly inhibited
[3H]dopamine uptake. Our results suggest that amphetamine-mediated dopamine release
through the plasmalemmal transporter is highly dependent on protein kinase C activity."
[Full Text]
Kramer
HK, Poblete JC, Azmitia EC.
3,4-Methylenedioxymethamphetamine ('Ecstasy')
promotes the translocation of protein kinase C (PKC): requirement of viable serotonin
nerve terminals.
Brain Res 1995 May 22;680(1-2):1-8
"The
metabolic effects of the neurotoxic, ring-substituted amphetamine 3,4-methylenedioxy-methamphetamine
(MDMA or 'Ecstasy') were examined in vivo. In this study, we focused on the ability
of MDMA to induce a translocation of the calcium and phospholipid-dependent protein
kinase C (PKC) from the cytosol to the cortical plasma membrane. Two injections
of MDMA (20 mg/kg; 10 h apart; s.c.) increased the density of membrane bound PKC
sites by 48.0% over saline treated animals without mediating a significant change
in ligand ([3H]phorbol 12,13 dibutyrate; [3H]PDBu) affinity. Longer drug treatments
(8 x 20 mg/kg) induced a lasting (up to 5 days post-treatment) increase in the
density of membrane-bound PKC. Prior destruction of cortical 5-HT nerve terminals
with p-chloroamphetamine (PCA) prevents this effect and suggests that viable 5-HT
uptake sites are essential for MDMA-induced PKC translocation. These results demonstrate
that MDMA-induced PKC translocation is mediated by viable cortical 5-HT nerve
terminals, and that prolonged kinase activation may contribute to MDMA-induced
serotonergic neurotoxicity." [Abstract]
[PDF] Kramer
HK, Poblete JC, Azmitia EC.
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 1998 Oct;19(4):265-77
"3,
4-methylenedioxymethamphetamine (MDMA or Ecstasy) is a substituted amphetamine
whose acute and long-term effects on the serotonin system are dependent on an
interaction with the 5-HT uptake transporter (SERT). Although much of the work
dedicated to the study of this compound has focused on its ability to release
monoamines, this drug has many important metabolic consequences on neurons and
glial cells. The identification of these physiological responses will help to
bridge the gap that exists in the information between the acute and neurotoxic
effects of amphetamines. Substituted amphetamines have the ability to produce
a long-term translocation of protein kinase C (PKC) in vivo, and this action may
be crucial to the development of serotonergic neurotoxicity. Our earlier results
suggested that PKC activation occurred through pre- and postsynaptic mechanisms.
Because the primary site of action of these drugs is the 5-HT transporter, we
now expand on our previous results and attempt to characterize MDMA's ability
to translocate PKC within cortical 5-HT nerve terminals. In synaptosomes, MDMA
produced a concentration-dependent increase in membrane-bound PKC (as measured
by 3H-phorbol 12, 13 dibutyrate, 3H-PDBu) bindings sites. This response was abolished
by cotreatment with the specific serotonin reuptake inhibitor (SSRI), fluoxetine,
but not by the 5-HT2A/2C antagonist, ketanserin. In contrast, full agonists to
5-HT1A and 5-HT2 receptors did not produce significant PKC translocation. MDMA-mediated
PKC translocation also requires the presence of extracellular calcium ions. Using
assay conditions where extracellular calcium was absent prevented in vitro activation
of PKC by MDMA. Prolonged PKC translocation has been hypothesized to contribute
to the calcium-dependent neurotoxicity produced by substituted amphetamines. In
addition, many physiological processes within 5-HT nerve terminals, including
5-HT reuptake and vesicular serotonin release, are susceptible to modification
by PKC-dependent protein phosphorylation. Our results suggest that prolonged activation
of PKC within the 5-HT nerve terminal may contribute to lasting changes in the
homeostatic function of 5-HT neurons, leading to the degeneration of specific
cellular elements after repeated MDMA exposure." [Abstract]
[PDF] Hansen,
J. Paul, Riddle, Evan L., Sandoval, Veronica, Brown, Jeffrey M., Gibb, James W.,
Hanson, Glen R., Fleckenstein, Annette E.
Methylenedioxymethamphetamine
Decreases Plasmalemmal and Vesicular Dopamine Transport: Mechanisms and Implications
for Neurotoxicity
J Pharmacol Exp Ther 2002 300: 1093-1100
"Administration
of a high-dose regimen of methamphetamine (METH) rapidly and profoundly decreases
plasmalemmal and vesicular dopamine (DA) transport in the striatum, as assessed
in synaptosomes and purified vesicles, respectively. To determine whether these
responses were common to other amphetamines of abuse, effects of methylenedioxymethamphetamine
(MDMA) on the plasmalemmal DA transporter (DAT) and vesicular monoamine transporter-2
(VMAT-2) were assessed. Similar to effects of METH reported previously, multiple
high-dose MDMA administrations rapidly (within 1 h) decreased plasmalemmal DA
uptake, as assessed ex vivo in synaptosomes prepared from treated rats. Unlike
effects of multiple METH injections, this deficit was reversed completely 24 h
after drug treatment. Also in contrast to effects of multiple METH injections,
1) MDMA caused little or no decrease in binding of the DAT ligand WIN35428, and
2) neither prevention of hyperthermia nor prior depletion of DA prevented the
MDMA-induced reduction in plasmalemmal DA transport. However, a role for phosphorylation
was suggested because pretreatment with protein kinase C inhibitors attenuated
the deficit caused by MDMA in an in vitro model system. In addition to affecting
DAT function, MDMA rapidly decreased vesicular DA transport as assessed in striatal
vesicles prepared from treated rats. Unlike effects of multiple METH injections
reported previously, this decrease partially recovered by 24 h after drug treatment.
Taken together, these results reveal several differences between effects of MDMA
and previously reported METH on DAT and VMAT-2; differences that may underlie
the dissimilar neurotoxic profile of these agents." [Full Text]
Giambalvo CT.
Mechanisms underlying the effects of amphetamine on particulate PKC activity.
Synapse. 2004 Feb;51(2):128-39.
Amphetamine stimulates particulate protein kinase C (PKC) activity that is associated with the outward-transport of dopamine (DA) (Giambalvo [2003] Synapse 49:125-133). This stimulatory effect requires intracellular calcium ([Ca](i)) and endogenous DA and when DA release is diminished, the inward-transport of amphetamine inhibits PKC activity. This study examines the mechanisms involved. It was found that synaptoneurosomes incubated with amphetamine showed a dose-dependent increase in phospholipase C and A(2) activities. Furthermore, pretreatments with the phospholipase C inhibitor D609 or the phospholipase A(2) inhibitors quinacrine or p-bromophenacylbromide attenuated the amphetamine-induced increase in PKC activity. This suggests that both phospholipases were essential for the amphetamine-induced increase in PKC activity. The Na/Ca antiporter was also involved, since pretreatment with amiloride or benzamil attenuated the amphetamine-induced increase in PKC activity. Since these drugs by themselves increased PKC activity, the return to basal activity after addition of amphetamine suggests that, in the absence of Na/Ca exchange, amphetamine had an inhibitory effect on PKC activity. This inhibitory effect might be due to the activation of phospholipase A(2) through an increase in intracellular pH induced by amphetamine. This was supported by the finding that pretreatment with dimethylamiloride, an inhibitor of the Na/H antiporter that increases intracellular [H(+)], attenuated the effects of amphetamine on PKC activity. Other drugs that decrease intracellular [H(+)] (ammonia, monensin) also inhibited PKC activity without Ca. In contrast to amphetamine, monensin had no effect on PKC activity with Ca. This could be related to its large differential effects on phospholipase A(2) vs. phospholipase C activity. Thus, the monensin-mediated decrease in PKC activity seen without Ca was partially attenuated by pretreatment with quinacrine. Furthermore, when Na/Ca antiporter was inhibited with benzamil, monensin inhibited PKC activity. These results suggest that amphetamine, as well as monensin, may have dual effects on PKC activity, a Ca-dependent stimulatory effect via phospholipase C, and an inhibitory effect via phospholipase A(2). [Abstract]
Schuldiner,
S, Steiner-Mordoch, S, Yelin, R, Wall, SC, Rudnick, G
Amphetamine
derivatives interact with both plasma membrane and secretory vesicle biogenic
amine transporters
Mol Pharmacol 1993 44: 1227-1231
"The
interaction of fenfluramine, 3,4-methylenedioxymethamphetamine (MDMA), and p-chloroamphetamine
(PCA) with the platelet plasma membrane serotonin transporter and the vesicular
amine transporter were studied using both transport and binding measurements.
Fenfluramine is apparently a substrate for the plasma membrane transporter, and
consequently inhibits both serotonin transport and imipramine binding. Moreover,
fenfluramine exchanges with internal [3H]serotonin in a plasma membrane transporter-mediated
reaction that requires NaCl and is blocked by imipramine. These properties are
similar to those of MDMA and PCA as previously described. In adrenal chromaffin
granule membrane vesicles containing the vesicular amine transporter, fenfluramine
inhibited serotonin transport and dissipated the transmembrane pH difference (delta
pH) that drives amine uptake. The use of [3H]reserpine-binding measurements to
determine drug interaction with the vesicular amine transporter allowed assessment
of the relative ability of MDMA, PCA, and fenfluramine to bind to the substrate
site of the vesicular transporter. These measurements permit a distinction between
inhibition of vesicular serotonin transport by directly blocking vesicular amine
transport and by dissipating delta pH. The results indicate that MDMA and fenfluramine
inhibit by both mechanisms but PCA dissipates delta pH without blocking vesicular
amine transport directly." [Abstract] Sammanda
Ramamoorthy, Elena Giovanetti, Yan Qian, and Randy D. Blakely
Phosphorylation
and Regulation of Antidepressant-sensitive Serotonin Transporters
J.
Biol. Chem. 273: 2458-2466, 1998.
"Antidepressant-sensitive serotonin
(5-hydroxytryptamine, 5HT) transporters (SERTs) are responsible for efficient
synaptic clearance of extracellular 5HT. Previously (Qian, Y., Galli, A., Ramamoorthy,
S., Risso, S., DeFelice, L. J., and Blakely, R. D. (1997) J. Neurosci. 17, 45-47),
we demonstrated that protein kinase (PKC)-linked pathways in transfected HEK-293
cells lead to the internalization of cell-surface human (h) SERT protein and a
reduction in 5HT uptake capacity. In the present study, we report that PKC activators
rapidly, and in a concentration-dependent manner, elevate the basal level of hSERT
phosphorylation 5-6-fold. Similarly, protein phosphatase (PP1/PP2A) inhibitors
down-regulate 5HT transport and significantly elevate hSERT 32P incorporation,
effects that are additive with those of PKC activators. Moreover, hSERT phosphorylation
induced by beta-phorbol 12-myristate 13-acetate is abolished selectively by the
PKC inhibitors staurosporine and bisindolylmaleimide I, whereas hSERT phosphorylation
induced by phosphatase inhibitors is insensitive to these agents at comparable
concentrations. Protein kinase A and protein kinase G activators fail to acutely
down-regulate 5HT uptake but significantly enhance hSERT phosphorylation. Basal
hSERT and okadaic acid-induced phosphorylation were insensitive to chelation of
intracellular calcium and Ca2+/calmodulin-dependent protein kinase inhibitors.
Together these results reveal hSERT to be a phosphoprotein whose phosphorylation
state is likely to be tightly controlled by multiple kinase and phosphatase pathways
that may also influence the transporter's regulated trafficking." [Full
Text]
Feuerstein TJ, Allgaier C, Hertting
G.
Possible involvement of protein kinase C (PKC) in the regulation
of electrically evoked serotonin (5-HT) release from rabbit hippocampal slices.
Eur
J Pharmacol 1987 Jul 23;139(3):267-72
"Protein kinase C (PKC)-activating
phorbol esters enhanced the electrically evoked 5-HT release from rabbit hippocampal
slices preincubated with [3H]5-HT. The release was diminished by polymyxin B,
an inhibitor of PKC. These results are compatible with a stimulatory effect of
PKC on the 5-HT release induced by action potentials. The mutual effects of PKC
affecting drugs on 5-HT release suggest a functional but not a competitive interaction.
The attenuation or the enhancement of effects of 5-HT autoreceptor ligands at
various 5-HT biophase concentrations found after PKC-affecting drugs are in line
with the view that autoreceptor-mediated events are not directly influenced by
the enzyme PKC." [Abstract] Sitte,
Harald H., Hiptmair, Birgit, Zwach, Julia, Pifl, Christian, Singer, Ernst A.,
Scholze, Petra
Quantitative Analysis of Inward and Outward Transport
Rates in Cells Stably Expressing the Cloned Human Serotonin Transporter: Inconsistencies
with the Hypothesis of Facilitated Exchange Diffusion
Mol
Pharmacol 2001 59: 1129-1137
"Quantitative aspects of inward and outward
transport of substrates by the human plasmalemmal serotonin transporter (hSERT)
were investigated. Uptake and superfusion experiments were performed on human
embryonic kidney 293 cells permanently expressing the hSERT using [(3)H]serotonin
(5-HT) and [(3)H]1-methyl-4-phenylpyridinium (MPP(+)) as substrates. Saturation
analyses rendered K(m) values of 0.60 and 17.0 microM for the uptake of [(3)H]5-HT
and [(3)H]MPP(+), respectively. Kinetic analysis of outward transport was performed
by prelabeling the cells with increasing concentrations of the two substrates
and exposing them to a saturating concentration of p-chloroamphetamine (PCA; 10
microM). Apparent K(m) values for PCA induced transport were 564 microM and about
7 mM intracellular [(3)H]5-HT and [(3)H]MPP(+), respectively. Lowering the extracellular
Na(+) concentrations in uptake and superfusion experiments revealed differential
effects on substrate transport: at 10 mM Na(+) the K(m) value for [(3)H]5-HT uptake
increased approximately 5-fold and the k value remained unchanged. The K(m) value
for [(3)H]MPP(+) uptake also increased, but the V(max) value was reduced by 50%.
When efflux was studied at saturating prelabeling conditions of both substrates,
PCA as well as unlabeled 5-HT and MPP(+) (all substances at saturating concentrations)
induced the same efflux at 10 mM and 120 mM Na(+). Thus, notwithstanding a 50%
reduction in the V(max) value of transport into the cell, MPP(+) was still able
to induce maximal outward transport of either substrate. Thus, hSERT-mediated
inward and outward transport seems to be independently modulated and may indicate
inconsistencies with the classical model of facilitated exchange diffusion."
[Full Text] Petra
Scholze, Lene Nørregaard, Ernst A. Singer, Michael Freissmuth, Ulrik Gether,
and Harald H. Sitte
The Role of Zinc Ions in Reverse Transport Mediated
by Monoamine Transporters
J. Biol. Chem. 277: 21505-21513,
2002.
"The human dopamine transporter (hDAT) contains an endogenous high
affinity Zn2+ binding site with three coordinating residues on its extracellular
face (His193, His375, and Glu396). Upon binding to this site, Zn2+ causes inhibition
of [3H]1-methyl-4-phenylpyridinium ([3H]MPP+) uptake. We investigated the effect
of Zn2+ on outward transport by superfusing hDAT-expressing HEK-293 cells preloaded
with [3H]MPP+. Although Zn2+ inhibited uptake, Zn2+ facilitated [3H]MPP+ release
induced by amphetamine, MPP+, or K+-induced depolarization specifically at hDAT
but not at the human serotonin and the norepinephrine transporter (hNET). Mutation
of the Zn2+ coordinating residue His(193) to Lys (the corresponding residue in
hNET) eliminated the effect of Zn2+ on efflux. Conversely, the reciprocal mutation
(K189H) conferred Zn2+ sensitivity to hNET. The intracellular [3H]MPP+ concentration
was varied to generate saturation isotherms; these showed that Zn2+ increased
V(max) for efflux (rather than K(M-Efflux-intracellular)). Thus, blockage of inward
transport by Zn2+ is not due to a simple inhibition of the transporter turnover
rate. The observations provide evidence against the model of facilitated exchange-diffusion
and support the concept that inward and outward transport represent discrete operational
modes of the transporter. In addition, they indicate a physiological role of Zn2+,
because Zn2+ also facilitated transport reversal of DAT in rat striatal slices."
[Full Text]
Khoshbouei
H, Wang H, Lechleiter JD, Javitch JA, Galli A.
Amphetamine-induced
DA efflux: A voltage sensitive and intracellular Na+-dependent mechanism.
J
Biol Chem 2003 Jan 29; [epub ahead of print]
"Amphetamine (AMPH) elicits
its behavioral effects by acting on the dopamine (DA) transporter (DAT) to induce
DA overflow into the synaptic cleft. Facilitated exchange diffusion is the classical
model used to describe AMPH-induced DA efflux. This model hypothesizes that AMPH-induced
DA efflux is mediated by DAT and results from transport of AMPH into the cell
followed by a counter movement of DA out to the extracellular compartment. To
further characterize the action of AMPH, we used the patch clamp technique in
the whole-cell configuration combined with amperometry on HEK-293 cells stably
transfected with the human DAT (DAT cells). In DAT cells, AMPH-induced DAT-mediated
currents were blocked by cocaine. We demonstrate that DA efflux mediated by DAT
is voltage dependent, electrogenic, and dependent on intracellular Na+ concentration
in the recording electrode. Intracellular Na+ fluorescence, as measured by confocal
microscopy using a Na+ sensitive dye, was enhanced by AMPH application. Furthermore,
the ability of AMPH to induce DA efflux was regulated by intracellular Na+ concentration
and correlated with the size of the DAT-mediated AMPH-induced ion flux across
the plasma membrane. In the absence of intracellular Na+, but in the presence
of high intracellular Cl-, AMPH-induced inward currents elicited DA efflux proportionally
to their dimension and duration. Thus, we propose that AMPH-induced DA efflux
depends on two-correlated transporter processes. First, AMPH binds to the DAT
and is transported, thereby causing an inward current. Second, because of this
AMPH-induced inward current, Na+ becomes more available intracellularly to the
DAT, thereby enhancing DAT-mediated reverse transport of DA." [Abstract/
Full Text] Connor CE, Kuczenski R.
Evidence
that amphetamine and Na+ gradient reversal increase striatal synaptosomal dopamine
synthesis through carrier-mediated efflux of dopamine.
Biochem
Pharmacol 1986 Sep 15;35(18):3123-30
"Amphetamine (AMPH) releases dopamine
(DA) from striatal synaptosomes and concomitantly increases DA synthesis. Since
AMPH may release DA through carrier-mediated diffusion via reversal of the DA
uptake system, the increase in DA synthesis might depend on a functioning uptake
carrier. Consistent with such a mechanism, the uptake inhibitors nomifensine (NMF)
and benztropine (BZT) completely prevented the AMPH-induced increase in DA synthesis
at concentrations known to inhibit DA uptake. Changes in the Na+ gradient across
the synaptosomal membrane also promote DA release, since DA and Na+ are cotransported
by the neuronal uptake carrier. Incubation of synaptosomes in medium containing
decreasing Na+ increased DA synthesis inversely proportional to Na+ over the range
128 to 20 mM. Similarly, incubations in the presence of 10(-4) M ouabain to inhibit
Na+, K+-ATPase and allow intracellular accumulation of Na+ also increased DA synthesis.
These changes in DA synthesis could also be prevented by BZT and were non-additive
with the AMPH-induced increase in DA synthesis. However, a concentration of ouabain
(10(-6) M) which by itself did not increase DA synthesis, and does not promote
DA release, potentiated the AMPH-induced increase in DA synthesis. Further, the
increased DA synthesis promoted by all three manipulations was only marginally
dependent on the presence of Ca2+ in the incubation medium. However, at 5 and
10 mM Na+, a second component of increased DA synthesis was observed which was
insensitive to BZT, but was prevented by Ca2+ removal. These results suggest that
the increase in DA synthesis, and presumably DA release promoted by AMPH, lowered
Na+, and ouabain, depend on the availability of the DA carrier at the internal
face of the neuronal membrane and the intracellular content of Na+. The second
component of increased DA synthesis which is evident at 5 and 10 mM Na+ is discussed
in terms of a possible Ca2+-mediated change in DA synthesis which is independent
of the DA carrier." [Abstract] Kantor,
Lana, Hewlett, G. H. Keikilani, Park, Yang Hae, Richardson-Burns, Sarah M., Mellon,
Mathew J., Gnegy, Margaret E.
Protein Kinase C and Intracellular
Calcium Are Required for Amphetamine-Mediated Dopamine Release via the Norepinephrine
Transporter in Undifferentiated PC12 Cells
J Pharmacol Exp
Ther 2001 297: 1016-1024
"The role of protein kinase C and intracellular
Ca(2+) on amphetamine-mediated dopamine release through the norepinephrine plasmalemmal
transporter in undifferentiated PC12 cells was investigated. The selective protein
kinase C inhibitor chelerythrine completely inhibited endogenous dopamine release
elicited by 1 microM amphetamine. Direct activation of protein kinase C increased
dopamine release in a Ca(2+)-insensitive, imipramine-sensitive manner and the
release was not additive with amphetamine. Exocytosis was not involved since these
events were not altered by either deletion of extracellular Ca(2+) or reserpine
pretreatment. Down-regulation of protein kinase C activity by long-term phorbol
ester treatment resulted in a dramatic decrease in amphetamine-mediated dopamine
release with no apparent effect on [(3)H]dopamine uptake. To more completely examine
a role for Ca(2+), intracellular Ca(2+) was chelated in the cells. Depletion of
intracellular Ca(2+) considerably decreased dopamine release in response to 1
microM amphetamine compared with vehicle-treated cells, but had no effect on the
[(3)H]dopamine uptake. Thus, our results suggest that amphetamine-mediated dopamine
release through the plasmalemmal norepinephrine transporter is highly dependent
on protein kinase C activity and intracellular but not extracellular Ca(2+). Furthermore,
protein kinase C and intracellular Ca(2+) appear to regulate [(3)H]dopamine inward
transport and amphetamine-mediated outward transport of dopamine independently
in PC12 cells." [Full
Text]
Koch S, Galloway MP.
MDMA
induced dopamine release in vivo: role of endogenous serotonin.
J
Neural Transm 1997;104(2-3):135-46
"Acting as a substrate at the serotonin
(5-HT) transporter, (+)-MDMA (3,4-methylenedioxymethamphetamine), is a potent
releaser of 5-HT and causes toxicity to 5-HT neurons after repeated exposure.
(+)-MDMA also releases dopamine (DA), although with less potency. Since we have
shown previously that the intrastriatal application of 5-HT facilities DA release,
it was hypothesized that increased release of striatal 5-HT after MDMA may influence
extracellular levels of DA. Using microdialysis in vivo, we found that (+)-MDMA
(4.7 mumol/kg, i.v.) administration increased extracellular striatal DA levels
to 501% of control (p < 0.01, n = 12). However, in the presence of fluoxetine
(14.4 mumol/kg, s.c.), which prevents (+)-MDMA effects on 5-HT release, the (+)-MDMA-induced
increase in DA was significantly less (to 375% of control, p < 0.05, vs. no
fluoxetine, n = 8). In vitro studies with striatal slices, to test drug selectivity,
showed that (+)-MDMA (0.3-3 microM) increased extracellular levels of both DA
and 5-HT in a dose-dependent manner. Fluoxetine (3 microM) completely blocked
the effects of (+)-MDMA on 5-HT release, but did not alter (+)-MDMA-induced DA
release in vitro. The selective DA transport inhibitor GBR-12909 (1 microM), blocked
(+)-MDMA's effect on DA release. It is concluded that 5-HT release after (+)-MDMA
treatment partially contributes to (+)-MDMA's effect on DA release in vivo."
[Abstract] Nash
JF, Brodkin J.
Microdialysis studies on 3,4-methylenedioxymethamphetamine-induced
dopamine release: effect of dopamine uptake inhibitors.
J
Pharmacol Exp Ther 1991 Nov;259(2):820-5
"The effect of dopamine (DA)
and serotonin (5-HT) uptake inhibitors on 3,4-methylenedioxymethamphetamine (MDMA)-induced
increase in DA efflux was studied using in vivo microdialysis. MDMA was infused
directly into the anterolateral striatum via the dialysis probe. The local administration
of MDMA produced a dose- and time-dependent increase in the extracellular concentration
of DA in the striatum. Peripheral administration of the DA uptake blockers, mazindol
(5 mg/kg, i.p.) or GBR 12909 (10 mg/kg, i.p.), produced a slight but significant
increase in the extracellular concentration of DA. Moreover, pretreatment with
either mazindol or GBR 12909 30 min before the infusion of MDMA (10 microM) significantly
attenuated the MDMA-induced increase in the extracellular concentration of DA.
Pretreatment with fluoxetine (10 mg/kg, i.p.), a 5-HT uptake blocker, 30 min before
the infusion of MDMA produced a slight but significant inhibition of MDMA-induced
increase in DA concentration. In contrast, pretreatment with the 5-HT2/1C antagonist,
ketanserin (3 mg/kg, i.p.), had no significant effect on the increase in DA concentration
produced by the local administration of MDMA. These data are suggestive that MDMA
increases the concentration of DA in the striatum, in part, via a carrier-mediated
mechanism which is largely independent of its effects on 5-HT release." [Abstract] Camarero
J, Sanchez V, O'Shea E, Green AR, Colado MI.
Studies, using in vivo
microdialysis, on the effect of the dopamine uptake inhibitor GBR 12909 on 3,4-methylenedioxymethamphetamine
('ecstasy')-induced dopamine release and free radical formation in the mouse striatum.
J
Neurochem 2002 Jun;81(5):961-72
"The present study examined the mechanisms
by which 3,4-methylenedioxymethamphetamine (MDMA) produces long-term neurotoxicity
of striatal dopamine neurones in mice and the protective action of the dopamine
uptake inhibitor GBR 12909. MDMA (30 mg/kg, i.p.), given three times at 3-h intervals,
produced a rapid increase in striatal dopamine release measured by in vivo microdialysis
(maximum increase to 380 +/- 64% of baseline). This increase was enhanced to 576
+/- 109% of baseline by GBR 12909 (10 mg/kg, i.p.) administered 30 min before
each dose of MDMA, supporting the contention that MDMA enters the terminal by
diffusion and not via the dopamine uptake site. This, in addition to the fact
that perfusion of the probe with a low Ca(2+) medium inhibited the MDMA-induced
increase in extracellular dopamine, indicates that the neurotransmitter may be
released by a Ca(2+) -dependent mechanism not related to the dopamine transporter.
MDMA (30 mg/kg x 3) increased the formation of 2,3-dihydroxybenzoic acid (2,3-DHBA)
from salicylic acid perfused through a probe implanted in the striatum, indicating
that MDMA increased free radical formation. GBR 12909 pre-treatment attenuated
the MDMA-induced increase in 2,3-DHBA formation by approximately 50%, but had
no significant intrinsic radical trapping activity. MDMA administration increased
lipid peroxidation in striatal synaptosomes, an effect reduced by approximately
60% by GBR 12909 pre-treatment. GBR 12909 did not modify the MDMA-induced changes
in body temperature. These data suggest that MDMA-induced toxicity of dopamine
neurones in mice results from free radical formation which in turn induces an
oxidative stress process. The data also indicate that the free radical formation
is probably not associated with the MDMA-induced dopamine release and that MDMA
does not induce dopamine release via an action at the dopamine transporter."
[Abstract] Metzger
RR, Hanson GR, Gibb JW, Fleckenstein AE.
3-4-Methylenedioxymethamphetamine-induced
acute changes in dopamine transporter function.
Eur J Pharmacol
1998 May 22;349(2-3):205-10
"The acute effects of the amphetamine designer
drug, 3,4-methylenedioxymethamphetamine (MDMA or 'ecstasy'), on dopamine transporter
function in rat striatum were investigated and compared to other psychostimulants
known to influence monoaminergic systems. A single MDMA injection (10-20 mg/kg;
s.c.) caused a dose-related decrease in [3H]dopamine uptake into striatal synaptosomes
prepared 1 h after MDMA administration. This rapid effect on [3H]dopamine uptake
returned to control levels 24 h after treatment. A single administration of other
amphetamine analogs, such as methamphetamine (15 mg/kg; s.c.), p-chloroamphetamine
(10 mg/kg; i.p.) or methcathinone (30 mg/kg; s.c.), also rapidly decreased striatal
[3H]dopamine uptake. In contrast, a single or multiple administrations of cocaine
(30 mg/kg; i.p.) had no effect on [3H]dopamine transport into striatal synaptosomes.
These changes in dopamine transporter activity by the amphetamine analogs may
occur via reactive oxygen species-mediated mechanisms." [Abstract]
Shankaran
M, Gudelsky GA.
Effect of 3,4-methylenedioxymethamphetamine
(MDMA) on hippocampal dopamine and serotonin.
Pharmacol
Biochem Behav 1998 Dec;61(4):361-6
"The 3,4-methylenedioxymethamphetamine
(MDMA)-induced increase in the extracellular concentration of dopamine and the
long-term depletion of 5-HT were studied in the hippocampus of the rat brain.
MDMA produced a dose-dependent increase in the extracellular concentration of
dopamine in the hippocampus, as well as in the striatum. The MDMA-induced increase
in the extracellular concentration of dopamine in the hippocampus, but not in
the striatum, was suppressed in rats treated with the norepinephrine uptake inhibitor,
desipramine, and in rats in which noradrenergic neurons in the hippocampus were
lesioned with DSP4 (N-(2- chloroethyl)-N-ethyl-2-bromo benzylamine). However,
the long-term depletion of 5-HT in the hippocampus produced by MDMA was unaltered
in desipramine-treated rats. These results are supportive of the view that the
MDMA-induced increase in the extracellular concentration of dopamine in the hippocampus
is the result of an enhanced release of dopamine from noradrenergic neurons. In
addition, the MDMA-induced depletion of 5-HT in the hippocampus appears not to
involve dopamine-initiated processes, because suppression of MDMA-induced dopamine
release did not attenuate the long-term depletion of 5-HT in the hippocampus."
[Abstract] Fitzgerald
JL, Reid JJ.
Effects of methylenedioxymethamphetamine
on the release of monoamines from rat brain slices.
Eur
J Pharmacol 1990 Nov 27;191(2):217-20
"The effects of 3,4-methylenedioxymethamphetamine
(MDMA) on monoamine release were investigated in superfused slices of rat striatum
and hippocampus. MDMA (10 microM) increased the resting release of radioactivity
from slices incubated in [3H]dopamine, [3H]5-hydroxytryptamine or [3H]noradrenaline.
These effects of MDMA (10 microM) were blocked by the neuronal uptake inhibitors,
cocaine (10 microM), fluoxetine (1 microM) and desmethylimipramine (1 microM),
respectively. MDMA (10 microM) enhanced the stimulation-induced efflux of radioactivity
from slices incubated in [3H]noradrenaline but not from slices incubated in [3H]5-hydroxytryptamine
or [3H]dopamine. These results demonstrate for the first time a direct noradrenaline-releasing
action of MDMA and differential effects of MDMA on the stimulation-induced release
of noradrenaline, dopamine and 5-hydroxytryptamine from rat superfused brain slices."
[Abstract] Kanthasamy
A, Sprague JE, Shotwell JR, Nichols DE.
Unilateral infusion of a
dopamine transporter antisense into the substantia nigra protects against MDMA-induced
serotonergic deficits in the ipsilateral striatum.
Neuroscience
2002;114(4):917-24
"The present study was designed to elucidate the consequences
of antisense oligonucleotide-mediated knockdown of striatal dopamine reuptake
transporters on 3,4-methylenedioxymethamphetamine (MDMA)-induced neurotoxicity.
Antisense oligonucleotide complementary to the mRNA translational start site of
the rat dopamine transporter was delivered by constant (7 days) intranigral infusion
with an osmotic minipump. Delivery of the antisense oligonucleotide by this method
resulted in a 70% reduction in the density of the dopamine transporter in the
ipsilateral striatum, as measured by [(3)H]mazindol binding. The effect of this
transporter knockdown on MDMA-induced serotonergic neurotoxicity was then examined.
MDMA (2x20 mg/kg, s.c., given 12 h apart) administered to control rats produced
hyperthermia following the first dose and led to a 45-50% reduction in striatal
serotonin, 5-hydroxyindoleacetic acid, and serotonin reuptake transporter density
1 week after the second dose. Conversely, in antisense-, but not missense-treated
rats, a significant attenuation of MDMA-induced neurotoxicity was observed only
in the ipsilateral striatum. The hyperthermic response elicited by MDMA was not
altered by prior administration of antisense. In vivo microdialysis revealed that
the antisense treatment attenuated MDMA-induced dopamine release in the ipsilateral
striatum.These results suggest that the dopamine transporter plays an essential
role in the neurodegeneration induced by MDMA, and provides additional support
for the hypothesis that extracellular dopamine is involved in the neurotoxic process,
at least in the striatum." [Abstract] Shankaran
M, Yamamoto BK, Gudelsky GA.
Mazindol attenuates the 3,4-methylenedioxymethamphetamine-induced
formation of hydroxyl radicals and long-term depletion of serotonin in the striatum.
J
Neurochem 1999 Jun;72(6):2516-22
"The formation of hydroxyl radicals following
the systemic administration of 3,4-methylenedioxymethamphetamine (MDMA) was studied
in the striatum of the rat by quantifying the stable adducts of salicylic acid
and D-phenylalanine, namely, 2,3-dihydroxybenzoic acid (2,3-DHBA) and p-tyrosine,
respectively. The repeated administration of MDMA produced a sustained increase
in the extracellular concentration of 2,3-DHBA and p-tyrosine, as well as dopamine.
The MDMA-induced increase in the extracellular concentration of both dopamine
and 2,3-DHBA was suppressed in rats treated with mazindol, a dopamine uptake inhibitor.
Mazindol also attenuated the long-term depletion of serotonin (5-HT) in the striatum
produced by MDMA without altering the acute hyperthermic response to MDMA. These
results are supportive of the view that MDMA produces a dopamine-dependent increase
in the formation of hydroxyl radicals in the striatum that may contribute to the
mechanism whereby MDMA produces a long-term depletion of brain 5-HT content."
[Abstract]
Al-Sahli
W, Ahmad H, Kheradmand F, Connolly C, Docherty JR.
Effects of methylenedioxymethamphetamine
on noradrenaline-evoked contractions of rat right ventricle and small mesenteric
artery.
Eur J Pharmacol 2001 Jun 22;422(1-3):169-74
"We
have compared the effects of methylenedioxymethamphetamine (MDMA) and cocaine
on contractions to noradrenaline in 1 Hz paced rat right ventricular strips, and
in rat small mesenteric artery and aorta. Noradrenaline increased the force of
contraction of 1 Hz paced ventricular strips with a pD(2) (-log EC(50)) of 5.64+/-0.07.
Both cocaine (10 microM) and MDMA (10 microM) significantly increased the potency
of noradrenaline to 6.31+/-0.11 and 6.42+/-0.13, respectively. However, in the
presence of cocaine (10 microM) which increased the potency of noradrenaline to
6.78+/-0.15, MDMA (10 microM) no longer increased the potency of noradrenaline
(pD(2) of 6.78+/-0.32). Likewise, following chemical sympathectomy, MDMA failed
to increase the potency of noradrenaline. The potency of the agonist isoprenaline,
which is not a substrate for the noradrenaline transporter, was not increased
by either cocaine or MDMA. In rat small mesenteric artery, but not aorta, MDMA
and cocaine significantly increased the potency of noradrenaline, but in the presence
of cocaine, MDMA had no further effect. Hence, MDMA shares with cocaine an ability
to potentiate the actions of noradrenaline, an action in the case of MDMA which
may involve competitive blockade of the noradrenaline transporter, rather than
simply displacement of noradrenaline. Since cocaine is linked to an increased
incidence of myocardial infarction, these results may have implications in terms
of cardiac morbidity of MDMA." [Abstract] Schuldiner
S, Steiner-Mordoch S, Yelin R, Wall SC, Rudnick G.
Amphetamine derivatives
interact with both plasma membrane and secretory vesicle biogenic amine transporters.
Mol
Pharmacol 1993 Dec;44(6):1227-31
"The interaction of fenfluramine, 3,4-methylenedioxymethamphetamine
(MDMA), and p-chloroamphetamine (PCA) with the platelet plasma membrane serotonin
transporter and the vesicular amine transporter were studied using both transport
and binding measurements. Fenfluramine is apparently a substrate for the plasma
membrane transporter, and consequently inhibits both serotonin transport and imipramine
binding. Moreover, fenfluramine exchanges with internal [3H]serotonin in a plasma
membrane transporter-mediated reaction that requires NaCl and is blocked by imipramine.
These properties are similar to those of MDMA and PCA as previously described.
In adrenal chromaffin granule membrane vesicles containing the vesicular amine
transporter, fenfluramine inhibited serotonin transport and dissipated the transmembrane
pH difference (delta pH) that drives amine uptake. The use of [3H]reserpine-binding
measurements to determine drug interaction with the vesicular amine transporter
allowed assessment of the relative ability of MDMA, PCA, and fenfluramine to bind
to the substrate site of the vesicular transporter. These measurements permit
a distinction between inhibition of vesicular serotonin transport by directly
blocking vesicular amine transport and by dissipating delta pH. The results indicate
that MDMA and fenfluramine inhibit by both mechanisms but PCA dissipates delta
pH without blocking vesicular amine transport directly." [Abstract] Kantor
L, Hewlett GH, Gnegy ME.
Enhanced amphetamine- and K+-mediated dopamine
release in rat striatum after repeated amphetamine: differential requirements
for Ca2+- and calmodulin-dependent phosphorylation and synaptic vesicles.
J
Neurosci 1999 May 15;19(10):3801-8
"After cessation of repeated, intermittent
amphetamine, we detected an emergent Ca2+-dependent component of amphetamine-induced
dopamine release and an increase in calmodulin and Ca2+- and calmodulin-dependent
protein kinase activity in rat striatum. This study examined the involvement of
calmodulin-dependent protein kinase II (CaM kinase II) and synaptic vesicles in
the enhanced Ca2+-dependent dopamine release in response to amphetamine or K+
in rat striatum. Rats were pretreated for 5 d with 2.5 mg/kg amphetamine or saline
and withdrawn from drug for 10 d. The selective CaM kinase II inhibitor KN-93
(1 microM), but not the inactive analog KN-92, attenuated the Ca2+-dependent amphetamine-mediated
dopamine release from amphetamine-pretreated rats but had no effect in saline-pretreated
controls. [3H]Dopamine uptake was unaltered by repeated amphetamine or KN-93 and
was Ca2+ independent. Striatal dopamine release stimulated by 50 mM KCl was enhanced
twofold after repeated amphetamine compared with that in saline controls but was
unaffected by KN-93. To examine the requirement for dopaminergic vesicles in the
Ca2+-dependent dopamine release, we administered reserpine to saline- and amphetamine-pretreated
rats 1 d before killing. Reserpine pretreatment did not affect amphetamine-mediated
dopamine release from either pretreatment group but completely ablated K+-mediated
dopamine release. Reserpine did not disrupt the ability of 1 microM KN-93 to block
the Ca2+-dependent amphetamine-mediated dopamine release from amphetamine-pretreated
rats. The results indicate that the enhanced dopamine release elicited by amphetamine
from chronically treated rats is dependent on Ca2+- and calmodulin-dependent phosphorylation
and is independent of vesicular dopamine storage. On the contrary, the enhanced
depolarization-mediated vesicular dopamine release is independent of Ca2+- and
calmodulin-dependent phosphorylation." [Abstract] |
Copeland BJ, Vogelsberg V, Neff NH, Hadjiconstantinou
M.
Protein kinase C activators decrease dopamine uptake into striatal
synaptosomes.
J Pharmacol Exp Ther 1996 Jun;277(3):1527-32
"Incubation
with either of the protein kinase C activators phorbol 12-myristate 13-acetate
(PMA) and sn-1,2 dioctanoylglycerol (DiC8) decreased the uptake of dopamine into
striatal synaptosomes, whereas the inactive phorbol ester 4 alpha-PMA had no effect.
Washout of PMA and DiC8 failed to reverse the decrease in uptake. Kinetic analysis
showed a decrease in the apparent V(max) for the transporter without changes in
the K(m). Neither PMA nor DiC8 affected mazindol binding to the dopamine transporter.
Preincubation with the protein kinase inhibitor staurosporine prevented the DiC8-induced
decrease of dopamine uptake. Furthermore, the protein phosphatase inhibitor okadaic
acid decreased dopamine uptake by itself and enhanced the DiC8-induced reduction
of uptake. These findings support a role for protein kinase C in modulating dopamine
transporter activity." [Abstract]
Cowell
RM, Kantor L, Hewlett GH, Frey KA, Gnegy ME.
Dopamine transporter
antagonists block phorbol ester-induced dopamine release and dopamine transporter
phosphorylation in striatal synaptosomes.
Eur J Pharmacol
2000 Feb 11;389(1):59-65
"We have reported that inhibition of protein
kinase C blocks the Ca(2+)-independent reverse transport of dopamine mediated
by amphetamine. In this study we investigated whether activation of protein kinase
C by 12-O-tetradecanoyl phorbol-13-acetate (TPA) would mediate dopamine release
through the plasmalemmal dopamine transporter. TPA, at 250 nM, increased the release
of dopamine from rat striatal slices and synaptosomes while the inactive phorbol
ester, 4alpha-phorbol, was ineffective. The TPA-mediated dopamine release was
independent of extracellular calcium and was blocked by a selective protein kinase
C inhibitor, Ro31-8220. The dopamine transporter antagonists, cocaine and GBR
12935 blocked the TPA-mediated dopamine release. In addition, cocaine blocked
TPA-mediated phosphorylation of the plasmalemmal dopamine transporter. These results
suggest that activation of protein kinase C results in reverse transport of dopamine
through the plasmalemmal dopamine transporter and the phosphorylated substrate
could be the dopamine transporter." [Abstract] Davis,
ME, Patrick, RL
Diacylglycerol-induced stimulation of neurotransmitter
release from rat brain striatal synaptosomes
J Neurochem
1990 54: 662-668
"These studies were undertaken to test the hypothesis
that alterations in phosphatidylinositol metabolism can modulate neurotransmitter
release in the central nervous system. The effects of 1,2- diacylglycerols (DAGs)
on dopamine release in the rat central nervous system were determined by measuring
dopamine release from rat striatal synaptosomes in response to two DAGs (sn-1,2-dioctanoylglycerol
and 1- oleoyl-2-acetylglycerol) that can activate protein kinase C and one DAG
(deoxydioctanoylglycerol) that does not activate this kinase. Dioctanoylglycerol
and 1-oleoyl-2-acetylglycerol, at a concentration of 50 micrograms/ml, stimulated
the release of labeled dopamine from striatal synaptosomes by 35-50 and 17%, respectively.
Dioctanoylglycerol-induced release was also demonstrated for endogenous dopamine.
In contrast, deoxydioctanoylglycerol (50 micrograms/ml) did not stimulate dopamine
release. Dioctanoylglycerol-induced dopamine release was independent of external
calcium concentration, indicating a utilization of internal calcium stores. Dioctanoylglycerol
(50 micrograms/ml) also produced a 38% increase in labeled serotonin release from
striatal synaptosomes. The addition of dioctanoylglycerol to the striatal supernatant
fraction increased protein kinase C activity. These results are consistent with
the concept that an increase in phosphatidylinositol metabolism can stimulate
neurotransmitter release in the central nervous system via an increase in DAG
concentration. The data suggest an involvement of protein kinase C in the DAG-induced
release, but other sites for DAG action are also possible." [Abstract]
Scholze,
Petra, Zwach, Julia, Kattinger, Alexandra, Pifl, Christian, Singer, Ernst A.,
Sitte, Harald H.
Transporter-Mediated Release: A Superfusion Study
on Human Embryonic Kidney Cells Stably Expressing the Human Serotonin Transporter
J
Pharmacol Exp Ther 2000 293: 870-878
"HEK 293 cells stably expressing
the human serotonin transporter (hSERT) were grown on coverslips, preincubated
with [(3)H]5-hydroxytryptamine (5-HT), and superfused. Substrates of the hSERT
[e.g., p-chloroamphetamine (PCA)], increased the basal efflux of [(3)H]5-HT in
a concentration-dependent manner. 5-HT reuptake blockers (e.g., imipramine, paroxetine)
also raised [(3)H]5-HT efflux, reaching approximately one-third of the maximal
effect of the hSERT substrates. In uptake experiments, both groups of substances
inhibited [(3)H]5-HT uptake. Using the low-affinity substrate [(3)H]N-methyl-4-phenylpyridinium
(MPP(+)) to label the cells in superfusion experiments, reuptake inhibitors failed
to enhance efflux. Similar results were obtained using human placental choriocarcinoma
(JAR) cells that constitutively express the hSERT at a low level. By contrast,
PCA raised [(3)H]MPP(+) efflux in both types of cells, and its effect was inhibited
by paroxetine. The addition of the Na(+),K(+)-ATPase inhibitor ouabain (100 microM)
to the superfusion buffer enhanced basal efflux of [(3)H]5-HT-loaded hSERT cells
by approximately 2-fold; the effect of PCA (10 microM) was strongly augmented
by ouabain, whereas the effect of imipramine was not. The Na(+)/H(+) ionophore
monensin (10 microM) also augmented the effect of PCA on efflux of [(3)H]5-HT
as well as on efflux of [(3)H]MPP(+). In [(3)H]5-HT-labeled cells, the combination
of imipramine and monensin raised [(3)H]5-HT efflux to a greater extent than either
of the two substances alone. In [(3)H]MPP(+)-labeled cells, imipramine had no
effect on its own and fully reversed the effect of monensin. The results suggest
that the [(3)H]5-HT efflux caused by uptake inhibitors is entirely due to interrupted
high-affinity reuptake, which is ongoing even under superfusion conditions."
[Full
Text]
Zahniser NR, Doolen S.
Chronic
and acute regulation of Na+/Cl- -dependent neurotransmitter transporters: drugs,
substrates, presynaptic receptors, and signaling systems.
Pharmacol
Ther 2001 Oct;92(1):21-55
"Na+/Cl- -dependent neurotransmitter transporters,
which constitute a gene superfamily, are crucial for limiting neurotransmitter
activity. Thus, it is critical to understand their regulation. This review focuses
primarily on the norepinephrine transporter, the dopamine transporter, the serotonin
transporter, and the gamma-aminobutyric acid transporter GAT1. Chronic administration
of drugs that alter neurotransmitter release or inhibit transporter activity can
produce persistent compensatory changes in brain transporter number and activity.
However, regulation has not been universally observed. Transient alterations in
norepinephrine transporter, dopamine transporter, serotonin transporter, and GAT1
function and/or number occur in response to more acute manipulations, including
membrane potential changes, substrate exposure, ethanol exposure, and presynaptic
receptor activation/inhibition. In many cases, acute regulation has been shown
to result from a rapid redistribution of the transporter between the cell surface
and intracellular sites. Second messenger systems involved in this rapid regulation
include protein kinases and phosphatases, of which protein kinase C has been the
best characterized. These signaling systems share the common characteristic of
altering maximal transport velocity and/or cell surface expression, consistent
with regulation of transporter trafficking. Although less well characterized,
arachidonic acid, reactive oxygen species, and nitric oxide also alter transporter
function. In addition to post-translational modifications, cytoskeleton interactions
and transporter oligomerization regulate transporter activity and trafficking.
Furthermore, promoter regions involved in transporter transcriptional regulation
have begun to be identified. Together, these findings suggest that Na+/Cl- -dependent
neurotransmitter transporters are regulated both long-term and in a more dynamic
manner, thereby providing several distinct mechanisms for altering synaptic neurotransmitter
concentrations and neurotransmission." [Abstract] Sandoval,
Veronica, Riddle, Evan L., Ugarte, Yvette V., Hanson, Glen R., Fleckenstein, Annette
E.
Methamphetamine-Induced Rapid and Reversible Changes in Dopamine
Transporter Function: An In Vitro Model
J. Neurosci. 2001
21: 1413-1419
"This laboratory has demonstrated that a single methamphetamine
(METH) injection rapidly and reversibly decreases the activity of the dopamine
transporter (DAT), as assessed ex vivo in synaptosomes prepared from treated rats.
This decrease does not occur because of residual drug introduced by the original
injection or nor is it associated with a change in binding of the DAT ligand WIN35428.
The purpose of this study was to elucidate the mechanism or mechanisms of this
METH effect by determining whether direct application of this stimulant to synaptosomes
causes changes in DAT similar to those observed ex vivo. Similar to the ex vivo
effect, incubation of striatal synaptosomes with METH decreased DAT activity,
but not WIN35428 binding: the effect on activity was not eliminated by repeated
washing of synaptosomes. Also, as observed ex vivo, incubation with 3,4-methylenedioxymethamphetamine,
but not cocaine or methylphenidate, caused a METH-like reduction in DAT function.
The rapid and reversible METH-induced diminution in DAT activity did not occur
because of a change in membrane potential, as assessed in vitro and ex vivo by
[3H]tetraphenylphosphonium accumulation. However, the METH-related decline in
DAT function may be attributed to phosphorylation because NPC15437, a protein
kinase C inhibitor, attenuated the METH-induced decline in DAT function. Similarities
between previously reported effects ex vivo of a single METH injection on serotonin
and norepinephrine transporter function and effects of direct METH application
in vitro were also found. Together, these data demonstrate that the in vitro incubation
model mimics the rapid and reversible effects observed after a single METH injection."
[Full Text]
Gulley
JM, Doolen S, Zahniser NR.
Brief, repeated exposure to substrates
down-regulates dopamine transporter function in Xenopus oocytes in vitro and rat
dorsal striatum in vivo.
J Neurochem 2002 Oct;83(2):400-11
"In
heterologous expression systems, dopamine transporter (DAT) cell-surface localization
is reduced after relatively prolonged exposure to d-amphetamine (AMPH) or dopamine
(DA), suggesting a role for substrate-mediated regulation of transporter function.
Here, we investigated whether brief, repeated periods of substrate exposure modulated
transporter function, first, in an in vitro model system and, second, in intact
rat brain. In human DAT-expressing Xenopus laevis oocytes, repeated exposure to
low micromolar concentrations of DA, AMPH or tyramine markedly reduced transport-mediated
currents. This functional down-regulation was attenuated by inclusion of a protein
kinase C (PKC) inhibitor and probably reflects DAT redistribution, as cell-surface
[3H]WIN 35 428 binding was significantly lower following DA exposure. High-speed
chronoamperometry was used to measure clearance of exogenously applied DA in dorsal
striatum (STR) and nucleus accumbens (NAc) of anesthetized rats. In STR, frequent
(every 2 min) applications of DA altered DA clearance parameters in a manner consistent
with profound down-regulation of DAT function. Similar changes were not observed
in NAc or after repeated vehicle (ascorbic acid) application. Together, our results
suggest that brief, repeated periods of substrate exposure lead to rapid down-regulation
of DAT activity and that this type of regulation can occur in vivo in STR, but
not NAc." [Abstract]
Crespi
D, Mennini T, Gobbi M.
Carrier-dependent and Ca(2+)-dependent 5-HT
and dopamine release induced by (+)-amphetamine, 3,4-methylendioxymethamphetamine,
p-chloroamphetamine and (+)-fenfluramine.
Br J Pharmacol
1997 Aug;121(8):1735-43
"1. The mechanism underlying 5-hydroxytryptamine
(5-HT) and/or dopamine release induced by (+)-amphetamine ((+)-Amph), 3,4-methylendioxymethamphetamine
(MDMA), p-chloroamphetamine (pCA) and (+)-fenfluramine ((+)-Fen) was investigated
in rat brain superfused synaptosomes preloaded with the 3H neurotransmitters.
2. Their rank order of potency for [3H]-5-HT-releasing activity was the same as
for inhibition of 5-HT uptake (pCA > or = MDMA > or = (+)-Fen > >
(+)-Amph). Similarly, their rank order as [3H]-dopamine releasers and dopamine
uptake inhibitors was the same ((+)-Amph > > pCA = MDMA > > (+)-Fen).
We also confirmed that the release induced by these compounds was prevented by
selective transporter inhibitors (indalpine or nomifensine). 3. [3H]-5HT and/or
[3H]-dopamine release induced by all these compounds was partially (31-80%), but
significantly Ca(2+)-dependent. Lack of extracellular Ca2+ did not alter uptake
mechanisms nor did it modify the carrier-dependent dopamine-induced [3H]-dopamine
release. (+)-Amph-induced [3H]-dopamine release and pCA- and MDMA-induced [3H]-5-HT
release were significantly inhibited by omega-agatoxin-IVA, a specific blocker
of P-type voltage-operated Ca(2+)-channels, similar to the previous results on
(+)-Fen-induced [3H]-5-HT release. 4. Methiothepin inhibited the Ca(2+)-dependent
component of (+)-Amph-induced [3H]-dopamine release with high potency (70 nM),
as previously found with (+)-Fen-induced [3H]-5-HT release. The inhibitory effect
of methiothepin was not due to its effects as a transporter inhibitor or Ca(2+)-channel
blocker and is unlikely to be due to its antagonist properties on 5-HT1/2, dopamine
or any other extracellular receptor. 5. These results indicate that the release
induced by these compounds is both 'carrier-mediated' and Ca(2+)-dependent (possibly
exocytotic-like), with the specific carrier allowing the amphetamines to enter
the synaptosome. The Ca(2+)-dependent release is mediated by Ca(2+)-influx (mainly
through P-type Ca(2+)-channels), possibly triggered by the drug interacting with
an unknown intracellular target, affected by methiothepin, common to both 5-HT
and dopamine synaptosomes." [Abstract]
Fournier F, Charnet P, Bourinet E, Vilbert C, Matifat
F, Charpentier G, Navarre P, Brule G, Marlot D.
Regulation by protein
kinase-C of putative P-type Ca channels expressed in Xenopus oocytes from cerebellar
mRNA.
FEBS Lett 1993 Feb 8;317(1-2):118-24
"Xenopus
oocytes injected with rat cerebellar mRNA expressed functional voltage-dependent
Ca channels detected as an inward Ba current (IBa). The pharmacological resistance
to dihydropyridines and omega-conotoxin together with the blockade obtained with
Agelenopsis aperta venom suggest that these channels could be somehow assimilated
to P-type Ca channels. The precise nature of the transplanted Ca channels was
assessed by hybrid-arrest experiments using a specific oligonucleotide antisense-derivated
from the recently cloned alpha 1-subunit of P channels (BI-1 clone). In addition,
we demonstrate that exogenous Ca channel activity was enhanced by two different
PKC activators (a phorbol ester and a structural analog to diacylglycerol). The
general electrophysiological and pharmacological properties of the stimulated
Ca channels remain unchanged. This potentiation induced by PKC activators is antagonized
by a PKC inhibitor (staurosporine) and by a monoclonal antibody directed against
PKC. It is concluded that P-type Ca channels are potentially regulated by PKC
phosphorylation and the functional relevance of this intracellular pathway is
discussed." [Abstract] Kimura
M, Yamanishi Y, Hanada T, Kagaya T, Kuwada M, Watanabe T, Katayama K, Nishizawa
Y.
Involvement of P-type calcium channels in high potassium-elicited
release of neurotransmitters from rat brain slices.
Neuroscience
1995 Jun;66(3):609-15
"Several types of voltage-dependent calcium channels
appear to occur in neurons, although coupling of the particular subtype of calcium
channels to the release of neurotransmitter has not been clearly understood. We
have examined the effects of subtype-specific inhibitors of the calcium channels
on depolarization-induced release of endogenous neurotransmitters from brain slices.
High potassium-induced release of glutamate and aspartate from hippocampal and
striatal slices was almost completely inhibited by a P-type channel blocker, omega-agatoxin
IVA. omega-Agatoxin IVA also completely inhibited the release of serotonin from
the hippocampal slices with almost the same potency as in the case of glutamate,
whereas the potency in blocking the release of serotonin and dopamine from striatal
slices was lower than that from the hippocampal slices. Another calcium channel
blocker, omega-agatoxin TK, that was recently found to block P-type channels with
very similar selectivity and potency to omega-agatoxin IVA, also inhibited the
release of amino acid transmitters and monoamines, though its potency was lower
than that of omega-agatoxin IVA. An N-type channel blocker, omega-conotoxin GVIA,
partially inhibited the neurotransmitter release, but an L-type channel blocker,
nifedipine was ineffective. We propose that the activation of P-type calcium channels
makes a major contribution to depolarization-elicited neurotransmitter release
in the CNS and that multiple P-type channels sensitive to omega-agatoxin IVA and
omega-agatoxin TK modulate the neurotransmitter release." [Abstract]
Dobrev D, Milde AS, Andreas K, Ravens U.
The
effects of verapamil and diltiazem on N-, P- and Q-type calcium channels mediating
dopamine release in rat striatum.
Br J Pharmacol 1999 May;127(2):576-82
"1.
The putative inhibitory effects of verapamil and diltiazem on neuronal non-L-type
Ca2+ channels were studied by investigating their effects on either K+- or veratridine-evoked
[3H]-dopamine ([3H]-DA) release in rat striatal slices. Involvement of N-, P-
and Q-type channels was identified by sensitivity of [3H]-DA release to omega-conotoxin
GVIA (omega-CTx-GVIA), omega-agatoxin IVA (omega-Aga-IVA) and omega-conotoxin
MVIIC (omega-CTx-MVIIC), respectively. 2. KCl (50 mM)-evoked [3H]-DA release was
abolished in the absence of Ca2+, and was insensitive to dihydropyridines (up
to 30 microM). It was significantly blocked by omega-CTx-GVIA (1 microM), omega-Aga-IVA
(30 nM) and was confirmed to be abolished by omega-CTx-MVIIC (3 microM), indicating
involvement of N-, P- and Q-type channel subtypes. 3. Verapamil and diltiazem
inhibited K+-evoked [3H]-DA release in a concentration-dependent manner. The inhibitory
effects of verapamil or diltiazem (each 30 microM) were fully additive to the
effect of omega-CTx-GVIA (1 microM), whereas co-application with omega-Aga-IVA
(30 nM) produced similar effects to those of omega-Aga-IVA alone. 4. As shown
previously, veratridine-evoked [3H]-DA release in Ca2+ containing medium exclusively
involves Q-type Ca2+ channels. Here, diltiazem (30 microM) did not inhibit veratridine-evoked
[3H]-DA release, whereas verapamil (30 microM) partially inhibited it, indicating
possible involvement of Q-type channels in verapamil-induced inhibition. However,
verapamil (30 microM) inhibited this release even in the absence of extracellular
Ca2+, suggesting that Na+ rather than Q-type Ca2+ channels are involved. 5. Taken
together, our results suggest that verapamil can block P- and at higher concentrations
possibly N- and Q-type Ca2+ channels linked to [3H]-DA release, whereas diltiazem
appears to block P-type Ca2+ channels only." [Abstract] Gnegy
ME, Hong P, Ferrell ST.
Phosphorylation of neuromodulin in rat striatum
after acute and repeated, intermittent amphetamine.
Brain
Res Mol Brain Res 1993 Dec;20(4):289-98
"Repeated, intermittent treatment
of rats with amphetamine results in a sensitization of locomotor and stereotyped
behaviors that is accompanied by an enhancement in stimulus-induced dopamine release.
Increased phosphorylation of the neural specific calmodulin-binding protein, neuromodulin
(GAP-43, B-50, F1) has been demonstrated in other forms of synaptic plasticity
and plays a role in neurotransmitter release. To determine whether neuromodulin
phosphorylation was altered during amphetamine sensitization, the in vivo phosphorylated
state of neuromodulin was examined in rat striatum in a post hoc phosphorylation
assay. Female, Holtzman rats received saline or 2.5 mg/kg amphetamine twice weekly
for 5 weeks. One week after the last dose of amphetamine, rats were challenged
with either 1 mg/kg or 2.5 mg/kg amphetamine or saline and the rats were sacrificed
30 min later. Purified synaptic plasma membranes were prepared in the presence
of EGTA and okadaic acid to inhibit dephosphorylation, and were subsequently phosphorylated
in the presence of purified protein kinase C and [gamma-32P]ATP. The protein kinase
C-mediated post hoc phosphorylation of neuromodulin was significantly reduced
in groups that received either acute or repeated amphetamine suggesting that neuromodulin
in those groups contained more endogenous phosphate. The acute, challenge dose
of amphetamine increased neuromodulin phosphorylation in the saline-treated controls
but not in the repeated amphetamine-pretreated group. Anti-neuromodulin immunoblots
showed no change in neuromodulin levels in any group. There was no significant
change in protein kinase C activity in any treatment group. To further investigate
the effect of acute amphetamine, the ability of amphetamine to alter neuromodulin
phosphorylation in 32Pi-preincubated Percoll-purified rat striatal synaptosomes
was examined. Amphetamine (10 microM) significantly increased phosphorylation
of a 53 kDa band that migrated with authentic neuromodulin in the synaptosomes
by 22% while 500 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) increased neuromodulin
phosphorylation by 45%. These data suggest that one injection of amphetamine can
increase neuromodulin phosphorylation in rat striatum and that this increase is
maintained for at least 1 week following a repeated, sensitizing regimen of amphetamine.
Since sensitization can be induced with one dose of amphetamine, it is possible
that enhanced neuromodulin phosphorylation could contribute to neurochemical events
leading to enhanced release of dopamine and/or behavioral sensitization."
[Abstract] Hens
JJ, De Wit M, Dekker LV, Boomsma F, Oestreicher AB, Margolis F, Gispen WH, De
Graan PN.
Studies on the role of B-50 (GAP-43) in the mechanism of
Ca(2+)-induced noradrenaline release: lack of involvement of protein kinase C
after the Ca2+ trigger.
J Neurochem 1993 Apr;60(4):1264-73
"The
involvement of B-50, protein kinase C (PKC), and PKC-mediated B-50 phosphorylation
in the mechanism of Ca(2+)-induced noradrenaline (NA) release was studied in highly
purified rat cerebrocortical synaptosomes permeated with streptolysin-O. Under
optimal permeation conditions, 12% of the total NA content (8.9 pmol of NA/mg
of synaptosomal protein) was released in a largely (> 60%) ATP-dependent manner
as a result of an elevation of the free Ca2+ concentration from 10(-8) to 10(-5)
M Ca2+. The Ca2+ sensitivity in the micromolar range is identical for [3H]NA and
endogenous NA release, indicating that Ca(2+)-induced [3H]NA release originates
from vesicular pools in noradrenergic synaptosomes. Ca(2+)-induced NA release
was inhibited by either N- or C-terminal-directed anti-B-50 antibodies, confirming
a role of B-50 in the process of exocytosis. In addition, both anti-B-50 antibodies
inhibited PKC-mediated B-50 phosphorylation with a similar difference in inhibitory
potency as observed for NA release. However, in a number of experiments, evidence
was obtained challenging a direct role of PKC and PKC-mediated B-50 phosphorylation
in Ca(2+)-induced NA release. PKC pseudosubstrate PKC19-36, which inhibited B-50
phosphorylation (IC50 value, 10(-5) M), failed to inhibit Ca(2+)-induced NA release,
even when added before the Ca2+ trigger. Similar results were obtained with PKC
inhibitor H-7, whereas polymyxin B inhibited B-50 phosphorylation as well as Ca(2+)-induced
NA release. Concerning the Ca2+ sensitivity, we demonstrate that PKC-mediated
B-50 phosphorylation is initiated at a slightly higher Ca2+ concentration than
NA release. Moreover, phorbol ester-induced PKC down-regulation was not paralleled
by a decrease in Ca(2+)-induced NA release from streptolysin-O-per |
