Peptides (v.27, #8)

Introductory note by Michael J. Kuhar (1911-1912).

The CART gene: Structure and regulation by Geraldina Dominguez (1913-1918).
CART peptides are important neuropeptides that are involved in a variety of physiologic processes. The regulation of the CART gene is critical since peptides are regulated and secreted in response to specific stimuli. CART mRNA must also be controlled in order to respond to specific stimuli such as psychostimulant drugs and leptin. The regulation of the CART gene is central to maintaining homeostasis of peptide production. The 5′ upstream region of the CART gene contains powerful regulatory elements that must be involved in transcriptional regulation via different signaling pathways. This review touches on several aspects related to CART gene regulation such as: (i) CART genomic structure, (ii) stimuli that alter CART mRNA levels, (iii) promoter characterization, (iv) role of the cAMP/PKA/CREB signal transduction pathway, and (v) role of the CART 5′ and 3′ ends in CART mRNA regulation. The goal of this review is to present current data so as to encourage further work in the field of CART gene regulation.
Keywords: CART gene; Promoter; CREB; CRE; PKA; Cocaine;

Cocaine- and amphetamine-regulated transcript (CART) peptides are expressed in several neuroendocrine tissues, including hypothalamus, pituitary, gut, adrenal and pancreas, and are involved in regulating important biological processes including feeding/appetite, drug reward and stress. CART is synthesized as larger, inactive peptide precursors (pro-CART) that require endoproteolytic processing to generate smaller, active forms. Prohormone/proprotein convertases (PCs), a family of calcium-dependent, serine endoproteases, have been shown to cleave many protein precursors in the regulated/constitutive secretory pathway to generate smaller fragments. In our previous studies, we have demonstrated the important roles of the two neuroendocrine-specific PCs, PC2 and PC1/3, in processing the two pro-CART isoforms, long (102aa) and short (89aa), to generate the bioactive CART peptides, I (55–102/42–89) and II (62–102/49–89) as well as the intermediate fragments, 10–89 and 33–102. Our subsequent studies have revealed the participation of another PC family member, PC5/6A (the soluble isoform of a widely expressed PC, PC5/6), in cleaving both precursor isoforms. We conclude that PC5/6A contributes to the normal efficient processing of pro-CART and is functionally more redundant with PC2 than PC1/3 in generating both CART I and II.
Keywords: CART (Cocaine- and amphetamine-regulated transcript); PC (Prohormone/proprotein convertase); Peptide precursor processing; Post-translational modification;

The activity of CART peptide fragments by Tomasz Dylag; Jolanta Kotlinska; Piotr Rafalski; Agnieszka Pachuta; Jerzy Silberring (1926-1933).
Cocaine- and amphetamine-regulated transcript (CART) peptides attracted much attention after the discovery that the level of CART mRNA is increased in rat striatum after acute administration of cocaine and amphetamine. The most widely investigated sequence is CART (55–102), whose roles were confirmed in modulation of various physiological processes such as feeding, energy expenditure, stress control, endocrine secretion, and reward. However, peptides other than (55–102) may be generated from the CART precursor as well.This review describes biological activity of peptides derived from the CART precursor in vivo, and of synthetic CART fragments that have not been found in the nature. In particular, the activity of CART (85–102) is described, whose ability to exert behavioral responses was confirmed by the observed attenuation of the expression of sensitization to morphine-induced hyperlocomotion. This fragment also decreased the number of escape jumps evoked by naloxone in morphine-addicted mice after intracerebroventricular administration.
Keywords: CART; Fragment; Cocaine; Amphetamine; Morphine; Dependence; Sensitization;

The CART receptors: Background and recent advances by Aleksandra Vicentic; Anita Lakatos; Douglas Jones (1934-1937).
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 CART's 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 [125I]-CART 61–102 was displaced only by active CART peptide but not by inactive CART fragments or several other unrelated peptides or drugs. The [125I]-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.
Keywords: CART signaling; GPCR; AtT20; ERK;

Ontogenetic expression of CART-peptides in the central nervous system and the periphery: A possible neurotrophic role? by P.Y. Risold; G. Bernard-Franchi; C. Collard; C. Jacquemard; A. La Roche; B. Griffond (1938-1941).
Little attention has been devoted to the expression of CART during development. However, a few studies in the central nervous system and periphery provide a clear indication that these peptides may play significant roles during histogenesis, and may have trophic actions.
Keywords: Development; Trophic factor; Rat; CART;

CART peptide diurnal variations in blood and brain by Aleksandra Vicentic (1942-1948).
The central role of CART peptide in feeding, drug abuse and stress has been widely researched however, CART's role in the peripheral system are less explored. CART peptide is present in a variety of peripheral tissues including sympathetic ganglion neurons, adrenal glands, gut, pancreas and blood. Studies that examined circulating CART demonstrated that the active fragment with a molecular weight of CART55-102 is present in the blood of rats and rhesus macaques. Interestingly, CART expression in these species exhibits a distinctive diurnal rhythm which correlates with the respective daily rhythms of corticosterone and feeding. In the rat, adrenalectomy significantly reduces blood CART levels and abolishes its daily rhythm while corticosterone replacement reinstates CART expression to control levels. In addition, direct administration of corticosterone significantly increases CART blood levels while administration of corticosterone synthesis blocker metyrapone, inhibits CART blood levels. These data suggest that the adrenal gland could be a source of blood CART and that glucocorticoids may play a role in the generation of CART's diurnal rhythm. Moreover, fuel availability may be important in the control of CART levels and its daily rhythm, since 24 h food restriction alters CART levels and abolishes its rhythm. In addition to blood, both CART peptide and mRNA exhibit food-dependent diurnal rhythm in discrete rat brain areas including the nucleus accumbens, amygdala and hypothalamus. Altogether, these findings suggest that CART is influenced by hypothalamic–pituitary–adrenal interactions and that it may play a role in multiple physiological processes possibly involving feeding, stress, reward and motivation.
Keywords: CART peptide; Blood; Diurnal rhythm;

Cocaine- and amphetamine-regulated transcript peptide and sympatho-adrenal axis by Siok L. Dun; G. Cristina Brailoiu; Jun Yang; Jaw Kang Chang; Nae J. Dun (1949-1955).
Cocaine- and amphetamine-regulated transcript peptide (CART) is constitutively expressed in discrete regions of the mammalian central and peripheral nervous system. Immunohistochemical studies reveal a well-defined network of CART-immunoreactive (irCART) neurons organized along the sympatho-adrenal axis. Sympathetic preganglionic neurons, but not parasympathetic preganglionic neurons, in the lateral horn area are CART-positive; which in turn innervate postganglionic neurons in the paravertebral and prevertebral sympathetic ganglia as well as the adrenal medulla. A population of chromaffin cells in the adrenal medulla is CART-positive; whereas, postganglionic neurons are not. Sympathetic preganglionic neurons themselves are contacted by irCART cell processes arising from neurons in the arcuate nucleus, the retrochiasmatic nucleus and the rostral ventrolateral medulla. Results from several recent studies suggest CART directly excites neurons along the sympathetic neural axis or indirectly by potentiating the action of glutamate on NMDA receptors, as evidenced by an elevation of blood pressure and heart rate following intracerebroventricular, intracisternal or intrathecal administration of the peptide to anesthetized rats or conscious rabbits.
Keywords: Cocaine; Sympatho-adrenal axis; Amphetamine; Sympathetic preganglionic neurons; Rostral ventrolateral medulla;

Cocaine and amphetamine regulated transcript (CART) and the stress response by Ersin O. Koylu; Burcu Balkan; Michael J. Kuhar; Sakire Pogun (1956-1969).
CART is expressed abundantly in the hypothalamic paraventricular nucleus and locus coeruleus, major corticotropin releasing factor (CRF) and noradrenaline sources, respectively. There is a bidirectional relation between CART and hypothalamo–pituitary–adrenal axis activity. CART stimulates CRF, adrenocorticotropic hormone and glucocorticoid secretion, whereas CRF and glucocorticoids increase the transcriptional activity of the CART gene; adrenalectomy declines CART expression in the hypothalamus. Stress exposure modulates CART expression in hypothalamus and amygdala in rat brain in a region and sex specific manner. CART may be a mediator peptide in the interaction between stress, drug abuse, and feeding. The review discusses the established role of CART as it relates to the stress response.
Keywords: CART; Stress; Corticotropin releasing factor; Glucocorticoids; Sex differences; HPA axis;

Anatomy of hypothalamic CART neurons by Niels Vrang (1970-1980).
Over the past 25 years the continuous discovery of novel neuropeptides has been a great aid in our understanding of central nervous system function. The neuropeptide CART was discovered in 1995 in a search for cocaine and amphetamine regulated transcripts in the striatum, but subsequently found to be expressed at much higher levels in the hypothalamus. Further studies on the distribution of both CART mRNA and CART immunoreactivity has added CART to the long list of neuropeptides expressed at high levels in several parts of the hypothalamus playing key roles in homeostasis and reproduction. Our extensive knowledge of hypothalamic function is due in great part to the high number of neuropeptides expressed in distinct hypothalamic cell groups, and naturally the discovery of CART led to myriad of papers examining possible roles played by CART peptides in different aspects of hypothalamic integration and reviewed elsewhere in this issue of Peptides. However, the rather widespread distribution of CART peptides in the brain certainly complicates the understanding of the role(s) played by this neurotransmitter and calls for careful interpretation of physiological/behavioral data. The aim of the present review is to focus attention on the rather complicated anatomy of the hypothalamic CART neurons, bearing in mind that a thorough understanding of brain function should be built on a solid anatomical foundation.
Keywords: In situ hybridization; Immunohistochemistry; Neuroanatomy; Cocaine-amphetamine regulated transcript; Review;

The role of CART in body weight homeostasis by Philip J. Larsen; Richard G. Hunter (1981-1986).
Cocaine–amphetamine regulated transcript (CART) was first identified as a complete mRNA transcript 10 years ago. Since then it has been demonstrated that CART is a peptide neurotransmitter which has a role in a number of physiological processes, including body weight homeostasis and energy balance, in a number of vertebrate species. Research to date has demonstrated a role for CART in the control of food intake at a number of levels within the brain of both animals and man.
Keywords: Cocaine–amphetamine regulated transcript; Homeostasis; Peptide; Feeding; Obesity;

CART peptide and the mesolimbic dopamine system by Kelly Philpot; Yoland Smith (1987-1992).
Over the past decade, CART peptide has been commonly associated with the rewarding and reinforcing properties of drugs of abuse and natural rewards such as food. The mesolimbic dopamine system is the predominant pathway involved in mediating reward and reinforcement. Many behavioral and neuroanatomical studies have been conducted in order to further elucidate the importance of CART-containing neurons within the mesolimbic dopamine system. This chapter will review the current knowledge of the localization, synaptic connectivity and neurochemical content of CART peptidecontaining neurons in nuclei of the mesolimbic reward pathway. These nuclei include the nucleus accumbens (NA), ventral midbrain, and the lateral hypothalamus (LH). In conclusion, an interconnected CART-containing loop between the NA, ventral midbrain and LH has evolved from these neuroanatomical studies that may have functional implications for CART peptide's involvement in reward and reinforcement.
Keywords: CART; Mesolimbic; Dopamine; Nucleus accumbens; Ventral tegmental area (VTA); Substantia nigra (SN); Lateral hypothalamus (LH);

The role of CART in the reward/reinforcing properties of psychostimulants by Jason N. Jaworski; Douglas C. Jones (1993-2004).
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.
Keywords: Psychostimulants; CART peptide; Neurotransmitter;

CART is a neuropeptide that appears to play an important role in a variety of physiological processes. The major research focus into the function of CART peptide has been on feeding behavior, modulation of mesolimbic dopamine, and actions of psychostimulant drugs. The neuroanatomic expression profile of CART does however suggest other functions as well, and its presence within the limbic system points to a possible role in emotionality. There are now several published reports which describe a new role for CART as a mediator of anxiety-like behaviors in rodents. This review will summarize these findings and speculate on the mechanisms by which CART might be involved in the modulation of these behaviors. We will also consider what future studies need to be done to further clarify the role of this peptide in anxiety.
Keywords: Cocaine- and amphetamine-regulated transcript; CART; Anxiety;

Cocaine- and amphetamine regulated transcript (CART) is a recently discovered anorexigenic peptide, widely expressed in the central nervous system. Included among presumed hypothalamic mediated functions of CART are inhibition of food intake, stimulation of energy expenditure and regulation of hypothalamic–pituitary axes. CART-immunoreactive (IR) axons densely innervate the majority of hypophysiotropic thyrotropin-releasing hormone-(TRH) containing neurons in the hypothalamic paraventricular nucleus (PVN) and establish asymmetric synaptic specializations with the TRH neurons. The CART-IR innervation of TRH neurons originates from at least two major sources: CART neurons in the arcuate nucleus that co-express the anorexigenic peptide, alpha-melanocyte-stimulating hormone (α-MSH), and adrenergic CART neurons in the medulla. Based on the origins of the CART innervation and potent stimulatory effects of CART on TRH gene expression of hypophysiotropic neurons, CART is suggested to be involved in the regulation of the hypothalamic–pituitary–thyroid (HPT) axis by different physiological stimuli. This regulatory control may contribute to the effects of fasting and cold exposure to reset the set point for feedback regulation of hypophysiotropic TRH gene expression and hence, affect circulating thyroid hormone levels. In addition, CART is present in the majority of hypophysiotropic TRH neurons and in TRH-containing axon terminals adjacent to the capillary vessels in the median eminence. While CART, alone, has no effect on the TSH and prolactin secretion from anterior pituitary cells, CART inhibits the stimulatory effect of TRH on prolactin secretion, but has no effect on TRH-induced increase of TSH release. Co-secretion of CART with TRH into the portal pituitary circulation, therefore, may have an important modulatory influence on the effect of TRH on pituitary hormone secretion.
Keywords: Cocaine- and amphetamine-regulated transcript (CART); Thyrotropin-releasing hormone (TRH); Hypothalamic–pituitary–thyroid axis; Hypothalamic paraventricular nucleus (PVN); Prolactin;

Intrathecal CART (55–102) attenuates hyperlagesia and allodynia in a mouse model of neuropathic but not inflammatory pain by M. Imad Damaj; Jianfeng Zheng; Billy R. Martin; Michael J. Kuhar (2019-2023).
CART peptides are found in brain and spinal cord areas involved in pain transmission. In the present study, we investigated the role of rat CART (55–102) in the modulation of chronic pain using models of chronic neuropathic (nerve injury model) and inflammatory (carrageenan test) pain models in the mouse after intrathecal administration. The results show that CART (55–102) was highly effective in reversing the hyperalgesia and allodynia signs of chronic neuropathic pain in a dose-related manner at doses (0.05–2 μg/mouse) that did not affect motor coordination of the animals. These effects lasted for at least 3 h after injection and were not blocked by naloxone, an opiate antagonist. Although CART (55–102) attenuated carrageenan-induced hyperalgesia, it failed to reduce the inflammation associated with this model. These results suggest the involvement of the CART peptides in the development of hyperalgesia and allodynia associated with neuropathic pain.
Keywords: CART; Neuropathic pain; Neuropeptides; Chronic pain;

CART in the enteric nervous system by Eva Ekblad (2024-2030).
The expression, distribution, origin, projections, chemical coding and functions of cocaine and amphetamine-regulated transcript (CART) in the gastro-intestinal tract are reviewed. CART is extensively expressed in the enteric nervous system. Except from being a possible modulator of NO induced intestinal relaxation CART does not seem to play any pivotal role in intestinal motility. Accumulating evidence suggest CART to be neuroprotective, involved in survival and maintenance of enteric neurons. CART expression increases in atrophic intestine thus suggesting a role of CART in intestinal adaptation. In rat antral mucosa CART is expressed in gastrin cells indicating a hormonal role of gastric CART.
Keywords: CART; Enteric nervous system; Intestinal adaptation; Enteric neuronal plasticity; Hirschsprung's disease;

CART is a novel islet regulatory peptide by Nils Wierup; Frank Sundler (2031-2036).
CART peptides have emerged as important islet regulators. CART is expressed both in islet endocrine cells and in parasympathetic and sensory nerves innervating the islets. In adult rats the intra-islet expression of CART is limited to the somatostatin producing δ-cells, while in adult mice CART is mainly expressed in nerve fibers. During development islet CART is upregulated; in rats in almost all types of islet endocrine cells, including the insulin-producing β-cells, and in mice mainly in the α-cells. This pattern of expression peaks around birth. CART is also expressed in human pancreatic nerves and in islet tumours where the expression level of CART may be related to the degree of differentiation of the tumour. Interestingly, in several rat models of type 2 diabetes CART expression is robustly upregulated in the β-cells, and is prominent during the phase of beta cell proliferation and hypertrophy. While CART inhibits glucose stimulated insulin secretion from rat islets it augments insulin secretion amplified by cAMP. Mice lacking CART, on the other hand, have islet dysfunction, and humans with a missense mutation in the cart gene are prone to develop type 2 diabetes. These data favor a role of CART in normal islet function and in the pathophysiology of type 2 diabetes.
Keywords: CART; Cocaine and amphetamine regulated transcript; Islet peptides; Insulin secretion; Type 2 diabetes; Neuropeptides; Islet development;

Studies of cocaine- and amphetamine-regulated transcript (CART) knockout mice by Mark Moffett; Lisa Stanek; Jill Harley; George Rogge; Mark Asnicar; Hansen Hsiung; Michael Kuhar (2037-2045).
CART (cocaine- and amphetamine-regulated transcript) peptides are neuropeptides expressed throughout the central nervous system and have been implicated in a variety of physiological processes. Research on the many physiological processes involving CART peptide have been somewhat limited by the lack of an identified CART antagonist. Development of CART peptide deficient mice has allowed scientists to further explore the many functions of CART peptide. This review briefly summarizes recent findings in the literature characterizing CART peptide deficient mice.