Search

Specific cocaine-amphetamine regulated transcript (CART) receptor binding has been reported in AtT20 and PC12 cells. In this study, receptor binding has been examined in primary cell cultures of several rat brain regions. Utilizing 125I-CART 61–102 as a ligand and CART 55–102 as a displacer, significant levels of specific binding compared with nonspecific binding were found in the nucleus accumbens. Specific CART binding was substantially lower in hippocampal, hypothalamic, and pituitary cells. The specific binding was saturable with a Kd of 1.43 ± 0.25 nM and a Bmax of 49.03 ± 2.33 fmol/mg protein. The binding was also highly specific for CART 55–102 as other inactive CART peptide fragments, unrelated peptides, γ-aminobutyric acid, or cocaine showed no affinity for the receptor at 10 μM concentrations. Furthermore, addition of the GTP analog, Gpp(NH)p, significantly decreased specific binding but addition of an ATP analog did not, suggesting that the receptor is a g-protein coupled receptor. This is the first demonstration of CART peptide receptor binding in primary cell cultures of the rat brain. Synapse 62:122–127, 2008. © 2007 Wiley-Liss, Inc.

In order to test for glucocorticoid regulation of CART in the arcuate nucleus, adrenalectomies (ADX) and hormone replacements (HRs) were carried out in groups of rats. CART mRNA levels were determined by in situ hybridization and CART peptide levels by immunocytochemistry. ADX caused a lowering of CART mRNA and peptides levels in the arcuate and this was reversed by HR. These results indicate a glucocorticoid regulation of CART in the arcuate. The regulation could be direct through an action of glucocorticoid receptors or indirectly through ADX-induced changes in leptin levels. These findings suggest a role for CART in the stress response.

CART peptides have been implicated in leptin-regulated feeding, reward and reinforcement, neurotropism, and other processes. In this Western blotting study, at least six different CART peptides varying from 4 to 14 kD were found in rat brain, pituitary, gut, and adrenal gland. The peptides may be processed differently in different tissues and one species found in the rat was not found in human hypothalamus. The higher molecular weight species are likely to include preproCART and proCART, while lower molecular weight peptides may be CART 55-102 and 62-102, physiologically active fragments.

RTI-113 [3β-(4-chlorophenyl)tropan-2β carboxylic acid phenyl ester hydrochloride], one of many phenyltropanes potent at and selective for DAT, inhibited self-administration of cocaine in rat at doses that did not alter responding maintained by food. The doses that inhibited cocaine intake produced significant levels of occupancy of DAT. Synapse 30:49–55, 1998. © 1998 Wiley-Liss, Inc.

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.

Cocaine and amphetamine regulated transcript (CART) encodes a novel brain-enriched protein whose features are reminiscent of a neurotransmitter propeptide. We have now localized CART peptide(s) in the gastrointestinal tract by immunohisto- chemical methods. Polyclonal antisera raised to CART peptide 106-129 stained neuronal cell bodies and fibers in rat ileum myenteric plexus-longitudinal muscle tissue prepara- tions. Electron microscopic analysis of thin sections showed immunopositive axon terminals in close apposition to CART-labelled and unlabelled neuronal cell bodies as well as to the longitudinal muscle. CART peptide-immunoreactive terminals contained numerous ovoid electron-lucent vesicles and a few dark-stained dense-core vesicles. Light microscopic double labelling studies revealed CART peptide immunoreactivity in a subpopulation of choline acetyltransferase (ChAT)-immunoreactive neurons. This com- bined light microscopic and ultrastructural examination of CART peptides in the gastrointestinal tract suggests a role of CART peptides as transmitters or neuromodula

While CART peptides have been implicated as novel, putative peptide neurotransmitters/cotransmitters, behavioral effects of these peptides have not yet been demonstrated. In this study, we show the first behavioral effect of CART peptides. Icv administration of CART peptide fragments inhibits feeding in rats. Moreover, injection of an antibody to CART peptide 82–103 stimulates feeding, suggesting that endogenous CART peptides exert an inhibitory tone on feeding. Injection of CART peptide 82–103 five min before NPY reduces the increase in feeding caused by injection of NPY alone. Also, in light microscopic immunohistochemical studies, NPY-positive varicosities were observed around CART peptide-positive cell bodies in the paraventricular nucleus of the hypothalamus. These data suggest functional interactions between CART peptides and NPY. These results indicate that CART peptides play a role in the control of food intake by the brain. Synapse 29:293–298, 1998. © 1998 Wiley-Liss, Inc.

CART (cocaine- and amphetamine-regulated transcript) peptides are proposed to play a role in the action of psychostimulants as neurotransmitters/neuromodulators. In the present study, we demonstrate that the shell of the nucleus accumbens, a brain structure involved in drug reinforcement, is densely innervated by a dense plexus of CART peptide-immunoreactive varicose fibers in register with immunoreactive perikarya in monkeys. At the electron microscopic level, varicosities appeared as immunoreactive axon terminals packed with round electron-lucent vesicles and a variable number of darkly stained dense-core vesicles that formed symmetric synapses with dendrites. These findings suggest that CART peptides may be a cotransmitter with gamma aminobutyric acid (GABA) in intrinsic axon collaterals of striatal projection neurons or interneurons in the primate nucleus accumbens.

Variously substituted phenyltropanes are proven as superb binding ligands for the dopamine transporter (DAT). In this study, we examine four N-substituted phenyltropanes which are derivatives of RTI-55 as in vivo binding ligands in mice. In this series, the methyl group on the nitrogen was replaced by a propyl (RTI-310), an allyl (RTI-311), a butyl (RTI-312), or a fluoropropyl (RTI-313) group. The in vitro binding potencies of these compounds at rat striatal DAT varied somewhat but were about 1 nM. While these compounds did not display marked selectivity for the dopamine transporter, they were more selective than RTI-55. Injection of the radiolabeled compound into mice resulted in striatal-to-cerebellar ratios that varied from about 4.5-6.5. The ratios peaked most rapidly for RTI-311 and RTI-313, at about 20 min. Pharmacological inhibition studies indicated that these compounds were binding to DATs in the striatum, as expected. These findings suggest that some compounds of this type may be excellent in vivo binding ligands for rapid imaging studies of the DAT.

[11C]WIN 35,428 (also designated [11C]CFT) is now being used in several positron emission tomography (PET) centers to image dopamine (DA) transporter sites in the mammalian brain. Whether and to what extent in vivo WIN 35,428 binding is influenced by intra- and extrasynaptic dopamine levels are largely unknown. The purpose of the present study was to evaluate the effects of various drugs, known to affect DA levels and release, on the binding of [3H]WIN 35,428 to central DA transporters in the mouse brain. D-Amphetamine, which releases DA from neurons and blocks the DA transporter directly, inhibited striatal [3H]WIN 35,428 binding in dose-dependent manner. Similarly, α-methyl-DL-p-tyrosine, an inhibitor of tyrosine hydroxylase, blocked [3H]WIN 35,428 binding, possibly via competitive inhibition by the metabolite p-hydroxyamphetamine. Specific binding of [3H]WIN 35,428 was insensitive to changes in synaptic DA levels caused by pretreatment of the animals with high doses of D2 receptor agonists (apomorphine, bromocriptine), antagonists (haloperidol) or the inhibitor of dopaminergic neuron firing γ-butyrolactone (GBL). High doses (>50 mg/kg) of L-DOPA (in combination with benserazide), however, reduced [3H]WIN 35,428 binding significantly, yet for a relatively short time (approximately 2.5 h). Chronic treatment with L-deprenyl elicited no changes in in vivo [3H]WIN 35,428 accumulation in the striatum. Neurotoxic damage of DA neurons caused by administration of high doses of amphetamine was detected in the striatum by a significant reduction in [3H]WIN 35,428 binding 7 days after cessation of amphetamine treatment. Thus, [3H]WIN 35,428 binding was only affected by neurotoxic loss of neurons, by administration of uptake inhibitors, or by some treatments which significantly elevate DA levels. Compounds which inhibit DA release or deplete DA acutely do not increase [3H]WIN 35,428 binding, suggesting that normal or “resting” levels of DA are not sufficient to alter [3H]WIN 35,428 binding in vivo. These findings are important for our understanding of the function and regulation of the DA transporter, as well as the in vivo binding of the radioligand [3H/11C]WIN 35,428. Moreover, they will be important for the interpretation of PET studies in which [11C]WIN 35,428 is used to assess the integrity of dopaminergic neurons. © 1996 Wiley-Liss, Inc.

In an extension of our previous work, an antibody directed against a peptide from the N-terminal region of DAT was used to localize specific dopamine transporter immunoreactivity (DAT-IR) in several regions of rat brain. Apparent axons and varicosities were found in the zona incerta, external layer of the median eminence, various nuclei of the amygdala, the cortex-amygdala transition zone, and in periglomerular regions in the olfactory bulb. Apparent stained neuronal perikarya and dendrites were observed in the arcuate nucleus and olfactory bulb. These regions are known to have dopaminergic neurons and innervations, although there was not a perfect correspondence between DAT-IR and the known distribution of dopaminergic neurons. A possible explanation is that different dopamine containing cell groups express different levels of DAT mRNA and protein, as we have previously shown. Also in the tuberoinfundibular neurons, for example, DAT-IR was preferentially localized to distal axons in the median eminence, suggesting intracellular compartmentation.

We have previously shown that withdrawal from repeated, intermittent infusions of cocaine in Lewis rats results in a long-lasting reduction in dopamine transporter levels in the nucleus accumbens. The reduction is dose-dependent, requires multiple injections as well as about a 10-day withdrawal period. In this investigation, we show that the decrease (34%) occurs in the shell rather than in the core of the nucleus accumbens, and that a second cycle of cocaine administration and withdrawal has no additional effect. Also, there were no changes in transporter binding in the caudate putamen, the olfactory tubercle or the ventral tegmental area. These results indicate that the limbic portions of the nucleus accumbens are involved in neurochemical adaptations during withdrawal from cocaine.

Cocaine analogs such as 3β-(4-iodophenyl)tropane-2β-carboxylic acid methyl ester (RTI-55 or βCIT) with a higher affinity for the dopamine transporter (DAT) may be potentially useful in interfering with cocaine's actions in brain. This study evaluates the time course of the effects of RTI-55 on cocaine binding in baboon brain using PET and [11C]cocaine. [11C]Cocaine binding was measured prior to, and 90 minutes, 24 hours, 4–5 days and 11–13 days after RTI-55 (0.3 mg/kg i.v.). Parallel studies with [3H]cocaine and RTI-55 (0.5 mg/kg i.v. or 2 mg/kg i.p.) were performed in the mouse. RTI-55 significalitly inhibited [11C]cocaine binding at 90 minutes and 24 hours after administration.The half-life for the clearance of RTI-55 from the DAT was estimated to be 2 to 3 days in the baboon brain. In the mouse brain, RTI-55 significantly inhibited [3H]cocaine binding at 60 and 180 minutes after administration and recovery was observed at 12 hours. These results document long-lasting inhibition of cocaine binding by RTI-55 and corroborate that binding kinetics of RTI-55 in striatum observed in imaging studies with [123I]RTI-55 represents binding to DATs. © 1995 Wiley-Liss, Inc.

3H-glutamate (GLU) uptake was measured in hippocampal synaptosomes from rat brain. Addition of sodium nitroprusside (SNP) (Sodium nitroferricyanide), a generator of nitric oxide (NO), produced a time-, temperature-, and dose-dependent inhibition of 3H-GLU uptake. The inhibition was due to changes in both Kd and Vmax of GLU uptake, and it was at least partially reversible upon washing. Addition of reduced hemoglobin (Hb), a substance that binds NO, prevented the SNP-induced depression of uptake. Potassium ferricyanide, a compound similar to SNP, did not cause a reduction in 3H-GLU uptake. Utilization of another generator of NO, S-nitroso-N-acetylpenicillamine (SNAP), produced similar results as did NO itself. Decreases in uptake were also observed in the striatum and cerebellum. Similar treatments did not consistently affect 3H-norepinephrine (NE) uptake, suggesting some selectivity in the NO effect. Thus, the observed inhibition of 3H-GLU uptake appears to be produced by NO, and it may represent a novel type of transynaptic retrograde regulation of transport. If found in vivo, inhibition of uptake activity could also be involved in the toxic effects of NO, the neurotoxicity of glutamate, and other potential neuronal changes associated with NO such as hippocampal long-term potentiation. © 1994 Wiley-Liss, Inc.1

[125I]RTI-55 was used tracer doses to label serotonin (5-HT) transporters in vivo in the mouse brain. Fluoxetine, paroxetine, and sertraline, potent antidepressants and selective inhibitors of serotonin transporter sites, were administered in various doses and at various times. The doses and times that result in significant binding of the drugs to transporters correspond to doses and times where they are reported to have physiological effects. Estimates of occupancy rate and duration of binding to serotonin transporters were made. The rate of occupancy of the 5-HT transporter site was fastest for sertraline, intermediate for paroxetine and slowest for fluoxetine. Similarly, the duration of occupancy was significantly shorter for sertraline and paroxetine (approximately 10 h) than for fluoxetine (approximately 50 h). The results indicate that in competition studies, [125I]RTI-55 can be used to identify doses of drugs that are physiologically effective, to determine their relative rate of occupancy, and most importantly, to measure the residency time on the central serotonin transporter in vivo.

[11C]WIN 35,428 was evaluated as a specific in vivo radioligand for the dopamine transporter site by PET scanning in nonhuman primates and humans. In studies with a baboon (Papio anubis), [11C]WIN 35,428 accumulated in brain regions containing dopamine transporters, i.e., the striata. This accumulation was Partially blocked by prior administration of (−)cocaine (4 mg/kg, i.v. ). Placement of a unilateral lesion of dopamine-containing nerve terminals with MPTP resulted in a unilateral reduction in [11C]WIN 35,428 accumulation in the striatum on the side of the lesion. Imaging of D2 dopamine receptors with [11C]NMSP in the same MPTP-treated animals showed much less reduction in the postsynaptic D2 dopamine receptors as compared to the much larger reduction in the dopamine transporters labeled with [11C]WIN 35,428. A total of ten normal human volunteers (five males and five females) with ages ranging from 19 to 81 years were studied. The caudate/cerebellar and putamen/cerebellar ratios ranged from 4.4 to 5.7 90 min after injection of the tracer. Preliminary kinetic modeling with arterial plasma sampling resulted in an average binding potential (K3/K4) of 4.98 in the caudate nucleus and 5.13 in putamen. To demonstrate in vivo blockade with dopamine reuptake inhibitors, two subjects received prior oral doses of 6 mg mazindol. Subject 5 had significant reductions of 29% in the caudate/cerebellar ratio at 90 min, 35% in the putamen/cerebellar ratio at 90 min, 45% in the caudate k3/k4 ratio from 6.7 to 3.7, and 46% in the putamen k3/k4 from 4.7 to 2.5. Subject 8 had significant reductions of 20% in both the caudate/cerebellar ratio and the putamen/cerebellar ratio at 90 min. During the human PET studies, a number of neuropsychological tests and physiological measurements were performed. No significant changes were found after administration of the [11C]WIN 35,428 alone. Taken together, these data indicate that [11C]WIN 35,428 is a promising radioligand for future studies of neuropsychiatric disorders that involve the dopamine transporter site. © 1993 Wiley-Liss, Inc.1

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.

[123I]RTI-55, an iodinated derivative of the cocaine analog 3β-phenyltropane-2β-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. Published Wiley-Liss, Inc.

n/a