Douglas C. Jones

CART peptides: regulators of body weight, reward and other functions

Over the past decade or so, CART (cocaine- and amphetamine-regulated transcript) peptides have emerged as major neurotransmitters and hormones. CART peptides are widely distributed in the CNS and are involved in regulating many processes, including food intake and the maintenance of body weight, reward and endocrine functions. Recent studies have produced a wealth of information about the location, regulation, processing and functions of CART peptides, but additional studies aimed at elucidating the physiological effects of the peptides and at characterizing the CART receptor(s) are needed to take advantage of possible therapeutic applications.

The metabolism and toxicity of quinones, quinonimines, quinone methides, and quinone-thioethers.

Quinones are ubiquitous in nature and constitute an important class of naturally occurring compounds found in plants, fungi and bacteria. Human exposure to quinones therefore occurs via the diet, but also clinically or via airborne pollutants. For example, the quinones of polycyclic aromatic hydrocarbons are prevalent as environmental contaminants and provide a major source of current human exposure to quinones. The inevitable human exposure to quinones, and the inherent reactivity of quinones, has stimulated substantial research on the chemistry and toxicology of these compounds. From a toxicological perspective, quinones possess two principal chemical properties that confer their reactivity in biological systems. Quinones are oxidants and electrophiles, and the relative contribution of these properties to quinone toxicity is influenced by chemical structure, in particular substituent effects. Modification to the quinone nucleus also influences quinone metabolism. This review will therefore focus on the differences in structure and metabolism of quinones, and how such differences influence quinone toxicology. Specific examples will be discussed to illustrate the diverse manner by which quinones interact with biological systems to initiate and propagate a toxic response.

Quantitative mapping of cytovhrome oxidase activity in the central auditory system of the gerbil: a study with calibrated activity standards and metal-intensified histochemistry

The objective was to obtain detailed topographic determinations of cytochrome oxidase activity in the gerbil central auditory system at the light microscopic level. Quantitative techniques were developed using (1) tissue standards calibrated to express histochemical measures as actual enzyme activity units, (2) densitometry and image analysis of histochemical reaction product formation, (3) spectrophotometry of cytochrome oxidase activity, and (4) a cobalt-intensified staining procedure compatible with autoradiography and other techniques requiring fresh-frozen brains without perfusion-fixation. Linear relationships between incubation time, section thickness, and activity of dissected brain regions, with their reaction product measured densitometrically were determined. Auditory structures with the high activities showed about 8 times the labeling intensity of the white matter or control sections inhibited with cyanide, glutaraldehyde, or heat. This indicated the high sensitivity of the method without loss of specificity. Specific activity for each of the 18 auditory structures measured were all above the units measured for whole brain homogenates, supporting the notion that basal levels of oxidative metabolism are greater for the auditory system. There was a progressive decrement in activity from brain stem to forebrain auditory structures. The more peripheral nuclei also showed a higher proportion of somatic as compared to neuropil reactivity. In contrast, auditory midbrain and thalamocortical regions were characterized primarily by neuropil reactivity. Comparison of intrinsic patterns of activity with morphological schemes to subdivide nuclei, showed a good correspondence with classical subdivisions derived from Golgi studies. The reported activities may provide a base of normative data in the gerbil for subsequent studies of central auditory functions. The method presented fulfilled established quantitative criteria and provided a more sensitive approach for regional mapping studies of brain cytochrome oxidase activity.

Dopamine-induced apoptosis is mediated by oxidative stress and Is enhanced by cyanide in differentiated PC12 cells.

Abstract: Dopamine (DA) oxidation and the generation of reactive oxygen species (ROS) may contribute to the degeneration of dopaminergic neurons underlying various neurological conditions. The present study demonstrates that DA‐induced cytotoxicity in differentiated PC12 cells is mediated by ROS and mitochondrial inhibition. Because cyanide induces parkinson‐like symptoms and is an inhibitor of the antioxidant system and mitochondrial function, cells were treated with KCN to study DA toxicity in an impaired neuronal system. Differentiated PC12 cells were exposed to DA, KCN, or a combination of the two for 12‐36 h. Lactate dehydrogenase (LDH) assays indicated that both DA (100‐500 μM) and KCN (100‐500 μM) induced a concentration‐ and time‐dependent cell death and that their combination produced an increase in cytotoxicity. Apoptotic death, measured by Hoechst dye and TUNEL (terminal deoxynucleotidyltransferase dUTP nick end‐labeling) staining, was also concentration‐ and time‐dependent for DA and KCN. DA plus KCN produced an increase in apoptosis, indicating that KCN, and thus an impaired system, enhances DA‐induced apoptosis. To study the mechanism(s) of DA toxicity, cells were pretreated with a series of compounds and incubated with DA (300 μM) and/or KCN (100 μM) for 24 h. Nomifensine, a DA reuptake inhibitor, rescued nearly 60‐70% of the cells from DA‐ and DA plus KCN‐induced apoptosis, suggesting that DA toxicity is in part mediated intracellularly. Pretreatment with antioxidants attenuated DA‐ and KCN‐induced apoptosis, indicating the involvement of oxidative species. Furthermore, buthionine sulfoximine, an inhibitor of glutathione synthesis, increased the apoptotic response, which was reversed when cells were pretreated with antioxidants. DA and DA plus KCN produced a significant increase in intracellular oxidant generation, supporting the involvement of oxidative stress in DA‐induced apoptosis. The nitric oxide synthase inhibitor NG‐nitro‐L‐arginine methyl ester and the peroxynitrite scavenger uric acid blocked apoptosis and oxidant production, indicating involvement of nitric oxide. These results suggest that DA neurotoxicity is enhanced under the conditions induced by cyanide and involves both ROS and nitric oxide‐mediated oxidative stress as an initiator of apoptosis.

The CART (cocaine- and amphetamine-regulated transcript) system in appetite and drug addiction.

CART (cocaine- and amphetamine-regulated transcript) peptides are neuromodulators that are involved in feeding, drug reward, stress, cardiovascular function, and bone remodeling. CART peptides are abundant but discretely distributed in the brain, pituitary and adrenal glands, pancreas, and gut. High expression of CART in discrete hypothalamic nuclei associated with feeding has led to behavioral and pharmacological studies that strongly support an anorectic action of CART in feeding. Subsequent studies on humans and transgenic animals provide additional evidence that CART is important in the regulation of appetite as mutations in the CART gene are linked to eating disorders, including obesity and anorexia. The expression of CART in the mesolimbic dopamine circuit has lead to functional studies demonstrating CARTs psychostimulant-like effects on locomotor activity and conditioned place preference in rats. These and other findings demonstrated that CART modulates mesolimbic dopamine systems and affects psychostimulant-induced reward and reinforcing behaviors. The link between CART and psychostimulants was substantiated by demonstrating alterations of the CART system in human cocaine addicts. CART seems to regulate the mesolimbic dopamine system, which serves as a common mechanism of action for both feeding and addiction. Indeed, recent studies that demonstrated CART projections from specific hypothalamic areas associated with feeding to specific mesolimbic areas linked to reward/motivation behaviors provide evidence that CART may be an important connection between food- and drug-related rewards. Given the enormous public health burden of both obesity and drug addiction, future studies exploring the pharmacotherapies targeting CART peptide represent an exciting and challenging research area.

The effects of environmental neurotoxicants on the dopaminergic system: A possible role in drug addiction

Humans are routinely exposed to a vast array of environmental neurotoxicants, including pesticides, endocrine disrupters, and heavy metals. The long-term consequences of exposure have become a major human health concern as research has indicated strong associations between neurotoxicants and a variety of dopamine-related neurological disorders. Developmental exposure to pesticides including paraquat, organochlorines, and rotenone produce alterations in the dopaminergic system and has been linked to neurodegenerative disorders, including Parkinsons disease. Endocrine disrupters such as Bisphenol A, mimic estrogenic activity and impact various dopaminergic processes to enhance mesolimbic dopamine activity resulting in hyperactivity, attention deficits, and a heightened sensitivity to drugs of abuse. A second class of endocrine disrupters, the polychlorinated biphenyls, may act directly on dopaminergic processes to disrupt the dopamine system and produce Parkinson-like symptoms. Exposure to the heavy metal lead enhances dopaminergic activity and has been associated with attention deficits, Alzheimers disease, and increased drug sensitivity. Manganese exposure, in contrast, results in dopamine deficiencies and Parkinson-like symptoms. Therefore, this commentary will discuss the effects and consequences that exposure to these three classes of environmental neurotoxicants have on the dopamine system and related behaviors and disorders. Finally, the recent hypothesis that exposure to environmental compounds which have effects on dopaminergic neurotransmission, including 2,4-dichlorophenoxyacetic acid, Bisphenol A, and multiple heavy metals, may potentiate drug-induced behaviors and increase the brains vulnerability to drug addiction will be discussed.

The role of metabolism in 3,4-(+)-methylenedioxyamphetamine and 3,4-(+)-methylenedioxymethamphetamine (ecstasy) toxicity.

Abstract: 3,4-Methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) are ring-substituted amphetamine derivatives with stimulant and hallucinogenic properties. The recreational use of these amphetamines, especially MDMA, is prevalent despite warnings of irreversible damage to the central nervous system. MDA and MDMA are primarily serotonergic neurotoxicants. Because (1) neither MDA nor MDMA produces neurotoxicity when injected directly into brain, (2) intracerebroventricular (ICV) administration of some major metabolites of MDA and MDMA fails to reproduce their neurotoxicity, (3) &agr;-methyldopamine (&agr;-MeDA) and N-methyl-&agr;-MeDA are metabolites of both MDA and MDMA, (4) &agr;-MeDA and N-methyl-&agr;-MeDA are readily oxidized to the corresponding ortho-quinones, which can undergo conjugation with glutathione (GSH), and (5) quinone thioethers exhibit a variety of toxicologic activities, we initiated studies on the potential role of thioether metabolites of &agr;-MeDA and N-methyl-&agr;-MeDA in the neurotoxicity of MDA and MDMA. Our studies have revealed that the thioether conjugates stimulate the acute release of serotonin, dopamine, and norepinephrine and produce a behavioral response commensurate with the “serotonin syndrome.” Direct injection of the conjugates into rat brain also produces long-term depletions in serotonin (5-HT) concentrations, elevations in GFAP expression, and activation of microglial cells. The data are consistent with the view that thioether metabolites of &agr;-MeDA and N-methyl-&agr;-MeDA contribute to the neurotoxicity of the parent amphetamines.

The role of CART in the reward/reinforcing properties of psychostimulants.

Cocaine- and amphetamine-regulated transcript (CART) peptides are putative neurotransmitters which appear to play a role in the rewarding and reinforcing effects of both natural (food) and unnatural (psychostimulants) stimuli. There is extensive anatomical, pharmacological, and behavioral evidence supporting the importance of CART peptides in psychostimulant, namely cocaine and amphetamine, abuse. For instance, CART mRNA and peptides are found in brain regions considered important in the reward and reinforcement of psychostimulants including the ventral tegmental area and the nucleus accumbens, which are part of the mesolimbic dopamine system. Consequently, in a pharmacological sense, CART peptides have been closely linked to the actions of mesolimbic dopamine. In addition, under certain conditions, psychostimulants alter CART mRNA and peptide levels. However, the exact conditions and mechanisms are unclear and may involve CART modulation by corticosterone and/or cyclic AMP response element binding protein (CREB). Finally, behavioral studies on CART and psychostimulants suggest a modulatory role for CART in the actions of psychostimulants as central administration of CART attenuates the behavioral effects of cocaine. This review discusses the anatomical, pharmacological, and behavioral evidence implicating a role for CART peptide in the rewarding and reinforcing properties of psychostimulants.

The CART receptors: background and recent advances.

Previous evidence obtained from several behavioral and biochemical studies suggested the existence of multiple CART receptors. However, identification of CART receptor binding has been largely unsuccessful until recently. The first evidence of CART signaling properties came from a study demonstrating that CART 55-102 inhibited voltage-dependent intracellular calcium signaling. More recent studies showed CART-induced dose- and time-dependent activation of extracellular signal-regulated kinase (ERK) 1 and 2 in AtT20 cell line. The activation of ERK was blocked by pertussis toxin but not genisten suggesting the involvement of Gi/o linked cascade in CARTs signaling properties in AtT20 cells. Shortly after these findings, the evidence of CART 61-102 specific binding was obtained from the same cell line. This study demonstrated that [(125)I]-CART 61-102 was displaced only by active CART peptide but not by inactive CART fragments or several other unrelated peptides or drugs. The [(125)I]-CART 61-102 binding was saturable and it had a high affinity for a single site in AtT20 cells. The binding was also dependent on time, pH, temperature and protein concentration. The average (+/-S.E.M.) B(max) and K(d) values were 101.4+/-8.8 fmol/mg protein and 21.9+/-8.0 pM, respectively. These data indicate the existence of specific CART receptor binding in AtT20 cells where CART signaling has been demonstrated. The identification of a receptor clone in these cells may help us elucidate CART receptors in other tissues. Because CART is implicated with several physiological functions including feeding, drug reward and stress, identification of a CART receptor would provide a novel target for the development of pharmacological tools and drugs for obesity and other disorders.

Regulation of CART mRNA in the rat nucleus accumbens via D3 dopamine receptors

A variety of studies indicate that CART in the nucleus accumbens (NAcc) is involved in the action of psychostimulants. In order to understand in more detail if and how dopamine is involved in the regulation of CART mRNA in the NAcc, the present studies of individual receptors were performed. The D1 agonist, dihydrexidine, and the D1 antagonist, SCH23,390, were administered separately and in combination to adult male rats; however, no changes were found in CART mRNA as measured by in situ hybridization. The D2/3 agonist, quinpirole, was administered either separately or in combination with the D2 selective antagonist, L741,626, or the D3 selective antagonist, GR103,691. Quinpirole produced a decrease in CART mRNA of up to 43%. This effect was blocked by pretreatment with the D3 antagonist GR103, 691, but not by the D2 antagonist, L741,626. CART peptide levels showed a similar decrement after acute quinpirole. CART mRNA levels in the NAcc of D3 mutant mice were found to be higher than that in wild-type animals, but treating the mutants with quinpirole failed to produce a decrease in CART expression like that observed in wild-type rodents. These findings demonstrate that CART is regulated by dopamine in the NAcc, at least partly by D3 dopamine receptors.