Jw Boja

Inhibition of striatal dopamine transporter activity by 17β-estradiol

Striatal synaptosomes from ovariectomized rats were prepared to examine the effect of 17beta-estradiol on [3H]dopamine uptake. Estradiol inhibited [3H]dopamine uptake in a dose-dependent manner, with an IC50 of 7.2 microM. Use of identical concentrations of progesterone had no effect on [3H]dopamine uptake. The effects of estradiol were exerted by decreasing the affinity of the transporter for dopamine, as revealed by a dose-dependent increase in the Km. The Km values for 0 (control), 10, and 100 microM estradiol were 108+/-11 258+/-44 and 415+/-40 nM, respectively, with each of the three concentrations tested being significantly different among each other. No statistically significant differences were obtained for the Vmax, with values for the three increasing doses being 9.2+/-0.8, 8.3+/-0.5 and 7.3+/-0.8 pmol/min per mg protein. These results demonstrate that estradiol, but not progesterone, inhibits striatal dopamine uptake by decreasing the affinity of the transporter for dopamine. Such a mechanism may serve as one of the bases for the modulatory effects of estradiol upon the nigrostriatal dopaminergic system.

Expression of a single dopamine transporter cDNA can confer two cocaine binding sites.

RAOIOLABELED cocaine analogs can bind to low and high affinity sites on striatal dopamine transporters (DAT). Recently, a cDNA encoding a rat brain dopamine transporter pDAT1 has been cloned. COS cells transfected with the pDAT1 in a eukaryotic expression vector express both a high (KD = 3.4 nM) and low affinity (KD = 163.6 nM) cocaine binding sites, suggesting that both sites are provided by a single gene product.

Selective dopamine transporter inhibition by cocaine analogs.

Several isopropyl and phenyl esters of 3 beta-(4-substituted phenyl) tropan-2 beta-carboxylic acid analogs of cocaine are relatively more potent and selective than cocaine and some other compounds in inhibiting dopamine uptake. These analogs can be used as binding ligands and as tools for elucidating the mechanisms of action of cocaine.

Stability of the stimulus properties of drugs over time

Three separate groups of rats were trained to discriminate the stimulus effects of either 600 mg/kg ethanol (n = 5), 0.8 mg/kg d-amphetamine (n = 8) or 1.0 mg/kg 1-(3-trifluoromethylphenyl)piperazine (TFMPP; n = 10). Once criterion performance was attained, each group was tested with various doses of the drug used in their training, thus allowing for calculations of dose-response curves and ED50 values. A second dose-response relationship was established at a later time, averaging over a year later, and this result was compared to the initial curve. In none of the three groups was there substantial change in the sensitivity of the rats to different doses of the drug used in training as indicated by similar ED50 values. These results suggest that the drug discrimination procedure is stable over a period of continuous training and testing.

CGS 10746B is able to attenuate the effects of amphetamine: Further evidence for dopaminergic mediation

Previous results indicate that agents which either decreases synthesis or block postsynaptic dopamine receptors will attenuate the discriminative stimulus produced by d-amphetamine. CGS 10746B has been reported to decrease dopamine release without changing its metabolism or occupying its receptors. In the present study, rats successfully trained to discriminate intraperitoneally administered (0.8 mg/kg) d-amphetamine in a two-lever, food-motivated operant task were observed to be unable to discriminate amphetamine when pretreated with 30 mg/kg CGS 10746B. This antagonism was shown to be dose-responsive and constitutes a third mechanism, i.e., dopamine release inhibition, that evidences the dopaminergic mediation of amphetamine in the discriminative paradigm. When both cathinone (0.8 mg/kg) and cocaine (10.0 mg/kg) were administered to the amphetamine-trained rats they each were recognized as amphetamine and are, thus, considered to generalize to the amphetamine discriminative stimulus. Coadministration of CGS 10746B and cathinone totally antagonized this generalization, whereas pretreatment with CGS 10746B prior to cocaine significantly reduced cocaines effects. These results implicate dopamine mechanisms in the discriminative stimulus properties of the psychostimulants amphetamine, cathinone and cocaine.

Behavioral effects of N-ethyl-3,4-methylenedioxyamphetamine (MDE; "EVE").

Eight male rats were trained to discriminate 2.0 mg/kg N-ethyl-3,4-methylenedioxyamphetamine (MDE) from its vehicle using a two-lever, food-motivated operant discrimination task. Once trained, the rats showed a dose-dependent decrease in discriminative accuracy following administration of decreased doses of MDE (ED50 = 0.75 mg/kg). Administration of 1.5 mg/kg 3,4-methylenedioxymethamphetamine (MDMA), a recently restricted Schedule I drug, produced 100% MDE-appropriate responding in the MDE-trained rats and decreased discriminative performance was similarly observed following lower doses of MDMA (ED50 = 0.62 mg/kg). The difference in relative potencies of MDE and MDMA in rats is reminiscent of those seen in human abusers who report effective oral psychotomimetic doses. Time-course data indicated that MDE has a fast onset, 100% drug-correct responding 10 min post-injection, and a peak effect between 10-20 min with declining effect at 60-120 min post-administration. These findings along with those of others show a pharmacological similarity between MDE and MDMA. Implications as to the future scheduling of MDE are discussed.

Multiple binding sites for [125I]RTI‐121 and other cocaine analogs in rat frontal cerebral cortex

In an effort to identify novel binding sites for cocaine and its analogs, we carried out binding studies with the high‐affinity and selective ligand [125I]RTI‐121 in rat frontal cortical tissue. Very low densities of binding sites were found. Saturation analysis revealed that the binding was to both high‐ and low‐affinity sites. Pharmacological competition studies were carried out with inhibitors of the dopamine, norepinephrine, and serotonin transporters. The various transporter inhibitors inhibited the binding of 15 pM [125I]RTI‐121 in a biphasic fashion following a two‐site binding model. The resultant data were complex and did not suggest a simple association with any single transporter. Correlational analysis supported the following hypothesis: [125I] RTI‐121 binds to known transporters and not to novel sites; these include dopamine, norepinephrine, and serotonin transporters. Immunoprecipitation of transporters photoaffinity labeled with [125]RTI‐82 and subsequent analysis of SDS‐page gels revealed the presence of authentic dopamine transporters in these samples; displacement of the photoaffinity label occurred with a typical dopamine transporter pharmacology. These data are compatible with the binding properties of RTI‐121 and the presence of several known transporters in the tissue studied. Synapse 30:9–17, 1998. Published 1998 Wiley‐Liss, Inc.

The antinociceptive effects of 3,4-methylenedioxymethamphetamine (MDMA) in the rat

The antinociceptive effects of MDMA and morphine were examined in rats using the tail-flick and hot-plate analgesiometric tests. MDMA, in the dose range of 1.5-6.0 mg/kg IP, produced a dose-dependent elevation in hot-plate latency, but did not elevate tail-flick latency. In contrast, morphine (2-8 mg/kg, IP) produced analgesia on both the tail-flick and hot-plate tests in a dose-dependent manner. Neither the opiate antagonist naltrexone nor the adrenoceptor antagonist phentolamine effectively attenuated MDMA-induced analgesia. Conversely, the serotonin antagonist methysergide significantly reversed the analgesic effects of MDMA on the hot-plate test. These findings suggest that the antinociceptive effects of MDMA are serotonergically mediated. Furthermore, the results verify earlier findings describing the test-specific effects of serotonin-induced pain modulation.

Norfenfluramine, the fenfluramine metabolite, provides stimulus control: Evidence for serotonergic mediation

Nine male rats were trained to discriminate 1.4 mg/kg norfenfluramine (NF) from its vehicle using a two-lever, food-motivated, operant discrimination task. Once trained, the rats showed a dose-dependent decrease in responding on the NF-correct lever following decreased doses of NF (ED50 = 0.71 mg/kg). Administration of 2.0 mg/kg fenfluramine (FEN) produced 100% responding on the NF-correct lever and decreasing doses of FEN, likewise, produced a dose-dependent decrease in responding on the NF-correct lever (ED50 = 1.30 mg/kg). Time-course data indicated that NF has a fast onset and a peak effect at 20-60 min after administration. Analysis of the time-course data provided a half-life of approximately 8 hr. In contrast, FEN did not show the rapid onset that was observed with NF. However, NF had a similar peak effect and half-life. These results indicate a pharmacological similarity between NF and FEN. However, the difference in onset of action suggests a possible difference between the parent drug and its metabolite. The serotonergic agonists mCPP, DOI, 5-MeODMT and LSD generalized to 1.4 mg/kg NF, whereas neither TFMPP nor 8-OHDPAT generalized to NF. The dopaminergic agonist AMPH also did not generalize to NF. The implications of these findings are discussed.

Brain reactive antibodies and the blood-brain barrier: Observations in aging rodents and the effects of peripheral kainic acid

This study was initiated to confirm the existence of brain-reactive autoantibodies and to determine if such antibodies have higher affinity for brain regions especially affected in Alzheimers disease. Serum collected from 90, 300, and 600 day old mice was incubated against brain tissues from these same mice, followed by incubation with fluorescently tagged rabbit antimouse IgG. No antibodies were present in the youngest serum, but considerable antibodies were present at 300 and, especially, at 600 days. Such antibodies were present in the blood vessels, but not in the brains of older animals. These antibodies, applied exogenously, labeled cells equally in all three ages of brains including most cortical and many other neurons, indicating that they are not neurotransmitter specific. In a further study, kainic acid or saline was administered peripherally to 15-month old rats. Kainic acid damaged the blood brain barrier and allowed the CNS entry of brain-reactive antibodies, especially into the subregions of hippocampus most damaged in Alzheimers.