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Biomedical subjects

M J Kuhar

Publications and source records attributed to M J Kuhar.

At least 19 recordsLinked to original sources
PubMed1992

Dopamine transport: pharmacological distinction between the synaptic membrane and the vesicular transporter in rat striatum.

The pharmacological properties of the monoamine transporters in the synaptic vesicles and of the dopamine transporters in the synaptic plasma membrane were compared. Tetrabenazine, an inhibitor of the vesicular transporter did not block ligand binding to the plasma membrane transporter. Various potent cocaine analogues and other compounds active at the plasma membrane transporter did not block ligand binding to the vesicular transporter. These data indicate pharmacological differences between the vesicular and synaptic membrane transporters.

Animals↗
PubMed1992

Microheterogeneity of dopamine transporters in rat striatum and nucleus accumbens.

Previously we have shown that the [125I]DEEP-labeled dopamine transporter from the rat nucleus accumbens has a higher apparent molecular weight than that from striatum. The present study confirms and extends these observations. Experiments with nucleus accumbens showed [125I]-DEEP to specifically bind to a protein with an apparent molecular weight of 76 kDa and with the pharmacological properties of the dopamine transporter. In exoglycosidase studies, treatment with neuraminidase, but not alpha-mannosidase, reduced the apparent molecular weight of the dopamine transporter from both the striatum and nucleus accumbens; however, a difference in the apparent molecular weight was still observed. N-Glycanase treatment, on the other hand, did reduce the apparent molecular weight of the dopamine transporters from the two regions to a similar value, approximately 56 kDa. In radioligand binding studies examining the effect of partial deglycosylation on striatal dopamine transporters, neuraminidase did not affect specific [3H]WIN 35,428 binding at 4 and 40 nM concentrations. In conclusion, the present study demonstrates that the difference in the apparent molecular weight of the dopamine transporter from these two regions is due to a difference in glycosylation and that the dopamine transporter from both regions contains similar amounts of sialic acid in their carbohydrate structure. Furthermore, the present data also indicate that the polypeptide portion of the dopamine transporter from both regions could be the same gene product.

Animals↗
PubMed1992

Probes for the cocaine receptor. Potentially irreversible ligands for the dopamine transporter.

Several potentially irreversible ligands (i.e., wash-resistant binding inhibitors) for the cocaine receptor site on the dopamine transporter, derived from (-)-cocaine or 3 beta-phenyltropan-2 beta-carboxylic acid methyl ester (WIN 35,065-2), were prepared and shown to produce wash-resistant inhibition of [3H]-3 beta-(p-fluorophenyl)tropan-2 beta-carboxylic acid methyl ester ([3H]WIN 35,428) binding. All the compounds prepared had the same absolute configuration as cocaine; they include analogues possessing chemically reactive groups such as the isothiocyanato and bromoacetamido as well as photoactive azido groups. The potentially irreversible ligands, as well as all the intermediates prepared in this study, were evaluated for their ability to inhibit the binding of [3H]WIN 35,428 in coincubation experiments. Of the potentially irreversible ligands, 3 beta-(p-chlorophenyl)tropan-2 beta-carboxylic acid 2-[p-(bromoacetamido)phenyl]ethyl ester (6c) had the highest apparent potency. The potentially irreversible ligands were also preincubated, and inhibition of [3H]WIN 35,428 binding was determined both before and after washing the ligand-exposed tissues. The most effective ligands in this regard were 3 beta-(3-iodo-4-azidophenyl)tropan-2 beta-carboxylic acid methyl ester (5) and 3 beta-(p-chlorophenyl)tropan-2 beta-carboxylic acid 2-(3-iodo-4-azidophenyl)ethyl ester (6d). The structure-activity relationships of these data are discussed.

Animals↗
PubMed1992

A cocaine analog and a GBR analog label the same protein in rat striatal membranes.

Because some evidence suggests that cocaine and GBR12935 bind to different sites, we utilized photoaffinity probes from both classes of compounds to see if they label the same protein. [125I]RTI-82 a cocaine analog, and [125I]DEEP, a GBR analog, labeled protein(s) showing the same molecular weight, a similar pharmacological profile and a similar sensitivity to neuraminidase.

Animals↗
PubMed1992

Molecular pharmacology of cocaine: a dopamine hypothesis and its implications.

The reinforcing properties of cocaine have been related to cocaine binding at the dopamine transporter in mesolimbocortical neurons. The molecular properties of the transporter have been studied in a number of laboratories. While this 'dopamine hypothesis' is strongly supported in animal studies of drug self-administration, the extent of its involvement in human drug dependence has not been fully elucidated.

Animals↗
PubMed1992

[123/125I]RTI-55, an in vivo label for the serotonin transporter.

[123I]RTI-55, an iodinated derivative of the cocaine analog 3 beta-phenyltropane-2 beta-carboxylic acid methyl ester, was evaluated as an agent for in vivo labeling of the serotonin transporter. Labeling of the precursor of RTI-55 with I-123 was efficient and yielded a high specific activity product. After intravenous injection of [123I]RTI-55 into rats, the tracer accumulated in regions with high densities of serotonin and dopamine uptake sites. The distribution of [123I]RTI-55 binding in areas rich in serotonin uptake sites correlated with [3H]serotonin uptake measured in vitro in the same regions. Specific [123I]RTI-55 binding to serotonin uptake sites was inhibited by paroxetine but not by GBR 12,909. Treatment of rats with neurotoxic doses of fenfluramine caused decreases of 66% (in the hypothalamus) to 83% (in the superior colliculi) of specific [125I]RTI-55 binding in all areas except in the striatum and the olfactory tubercles (regions rich in dopamine transporters). These results indicate that [123/125I]RTI-55 binds, although not selectively, to the serotonin transporter in vivo. Furthermore, they suggest that [123I]RTI-55 holds promise as a SPECT imaging agent for the study of the serotonin transporter in humans in health and disease.

Animals↗
PubMed1992

High-affinity binding of [125I]RTI-55 to dopamine and serotonin transporters in rat brain.

RTI-55 (3 beta-(4-iodophenyl)tropan-2 beta-carboxylic acid methyl ester), one of the most potent inhibitors of dopamine uptake reported to date, was radioiodinated and tested as a probe for the cocaine receptor in Sprague-Dawley rat brain. Saturation and kinetic studies in the striatum revealed that [125I]RTI-55 bound to both a high- and low-affinity site. The Kd for the high-affinity site was 0.2 nM, while the Kd for the low-affinity site was 5.8 nM. The corresponding number of binding sites in the striatum was 37 and 415 pmol/g protein. The pharmacological profile of specific [125I]RTI-55 binding in the striatum was consistent with that of the dopamine transporter. Additionally, [125I]RTI-55 was found to bind with high affinity to the cerebral cortex. Scatchard analysis revealed a single high-affinity component of 0.2 nM with a density of 2.5 pmol/g protein. The pharmacological profile demonstrated by [125I]RTI-55 in the cerebral cortex matched that of the serotonin transporter. Autoradiographic analysis of sagittal brain sections with [125I]RTI-55 binding was consistent with these findings. Specific binding of [125I]RTI-55 was blocked by dopamine uptake inhibitors in areas rich in dopaminergic nerve terminals. Conversely, serotonin uptake inhibitors blocked the binding of [125I]RTI-55 in brain areas rich in serotonergic neurons. These results demonstrate that [125I]RTI-55 may be a very useful ligand for the dopamine and serotonin transporters.

Animals↗
PubMed1992

In vivo binding of [125I]RTI-55 to dopamine transporters: pharmacology and regional distribution with autoradiography.

Previous studies have demonstrated that para-substituted WIN 35,065-2 analogs of cocaine show high binding affinity for dopamine uptake sites both in vitro and in vivo, and inhibit DA uptake in vitro. These analogs also produce potent cocaine-like behavioral effects in various procedures. The purpose of the present studies was to evaluate the iodinated WIN 35,065-2 analog [125I]RTI-55 as an in vivo ligand for the DA transporter. Following intravenous injection in mice, [125I]RTI-55 showed highest accumulation in areas with high densities of dopamine uptake sites. Light microscopic autoradiography was used to examine binding with higher resolution. Displacement studies demonstrated that [125I]RTI-55 binding in dopamine containing regions, striatum and olfactory tubercles, was saturable and inhibited by other cocaine analogs. GBR 12909 and WIN 35,428 significantly inhibited [125I]RTI-55 binding in striatum, while paroxetine significantly inhibited hypothalamic binding but had little effect in striatum. The latter finding suggests that [125I]RTI-55 also binds to the serotonin transporter. Haloperidol had no effect on [125I]RTI-55 binding in any brain region measured. In addition, treatment of animals with the dopamine neurotoxin MPTP caused significant reductions in striatal [125I]RTI-55 binding. The results of these studies indicate that [125I]RTI-55 binds primarily to the dopamine transporter in the mouse striatum in vivo.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1992

Molecular mechanisms associated with cocaine effects. Possible relationships with effects of ethanol.

Cocaine has been shown to be a highly addictive and toxic drug. It produces these effects and a variety of other physiological and behavioral effects through its interactions with several distinct central nervous system receptor sites. We present the results of a series of studies that utilized multiple site analyses to elucidate which cocaine binding sites influence the reinforcing and toxic effects of cocaine and with what proportion of influence. The nature of cocaine interactions with monoamine transporters is also discussed, especially with the dopamine transporter, which has been shown to be the cocaine binding site that is primarily associated with the reinforcing effects of cocaine. We also provide evidence that vulnerability to both the toxic and addictive effects of cocaine may be significantly influenced by genetic differences in both humans and animals. In view of the fact that cocaine is commonly abused in a polydrug situation, we present the results of both behavioral and biochemical experiments which suggest that common biochemical pathways may mediate the reinforcing or addictive properties of drugs of abuse. Finally, we discuss research on the biochemical mechanisms associated with effects of ethanol, particularly those which may also influence cocaine self-administration and speculate on pharmacotherapeutic strategies for concurrent abuse of cocaine and ethanol.

Alcoholism↗
PubMed1992

2 beta-substituted analogues of cocaine. Synthesis and inhibition of binding to the cocaine receptor.

The potencies of a series of 2 beta-substituted cocaine analogues to displace [3H]-3 beta-(p-fluorophenyl)tropane-2 beta-carboxylic acid methyl ester binding in rat striatal membranes demonstrate the requirement for a 2 beta-substituent with two hydrogen-bond acceptors. The insensitivity of the ester moiety to steric and electronic factors suggests its modification to provide site-specific irreversible ligands.

Analgesics↗
PubMed1992

N-modified analogues of cocaine: synthesis and inhibition of binding to the cocaine receptor.

Cocaine methiodide (2), N-norcocaine (1b), N-benzyl-N-norcocaine (1c), and N-nor-N-acetylcocaine (1d) were synthesized and evaluated for their ability to inhibit binding of [3H]-3 beta-(4-fluorophenyl)tropane-2 beta-carboxylic acid methyl ester (WIN 35,428) to the cocaine receptor. The study showed that removal of the N-methyl group to give 1b, or replacement with the larger N-benzyl group to give 1c, has a relatively small effect on binding potency. In contrast, replacement of the N-methyl group by the acetyl moiety to give 1d, or the addition of a methyl group to give 2, reduces affinity for the receptor by a large factor. In order to gain preliminary information concerning the importance of the nitrogen location on the tropane ring system, the receptor binding affinity of 8-methyl-8-azabicyclo[3.2.1]octan-3 beta-ol benzoate (5, beta-tropacocaine) was compared to that of the isomeric 6-methyl-6-azabicyclo[3.2.1]octan-3 beta-ol benzoate (4d). The fact that both compounds have similar binding affinities for the cocaine receptor suggests that 3 beta-(benzoyloxy)-6-methyl-6-azabicyclo[3.2.1] octane-2-carboxylic acid methyl ester, which is isomeric with cocaine, may possess binding potency similar to cocaine.

Analgesics↗
PubMed1992

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

Radiolabeled 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.

Animals↗
PubMed1992

Dopamine transporter imaging with novel, selective cocaine analogs.

RTI-121 and RTI-122 are 3 beta-substituted phenyltropane analogs of cocaine that have high, selective binding affinity for dopamine transporters. [123I]RTI-121 and [123I]RTI-122 bind to dopamine transporters in vivo after intravenous administration and permit imaging of the transporters.

Animals↗
PubMed1992

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.

Animals↗
PubMed1992

Behavioral effects of novel cocaine analogs: a comparison with in vivo receptor binding potency.

Several novel cocaine analogs, previously shown to be very potent in in vitro binding studies, have been examined for their stimulatory effects on locomotor activity and for their ability to displace [3H]WIN 35,428 binding in vivo in mice. These compounds, like WIN 35,428, lack an ester link between the phenyl group and the tropane ring and have para-substitutions on the phenyl ring. They were much more potent than (-)-cocaine in producing increases in locomotor activity. In addition, they were more potent than (-)-cocaine in inhibiting [3H]WIN 35,428 binding in vivo in mouse striatum. Thus, these compounds demonstrate similar high potency in behavioral tests and in receptor binding assays, both in vivo and in vitro. Results support the hypothesis of a relationship between binding at the dopamine transporter and the behavioral effects of cocaine-like drugs. Further, assuming that maximal occupancy occurs with total displacement of [3H]WIN 35,428 binding in vivo, the data suggest that maximal locomotor effects occur with near total occupancy of transporter binding sites.

Animals↗