Intrathecal CART (55-102) attenuates hyperlagesia and allodynia in a mouse model of neuropathic but not inflammatory pain

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 microg/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.     

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 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 [(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.     

CART (85-102)-inhibition of psychostimulant-induced hyperlocomotion: importance of cyclization

Synthetic derivative of C-terminal fragment of CART (55–102) with reduced thiol groups, [Abu^86,94]CART (85–102)_red, given together with amphetamine (5 mg/kg, s.c.) or cocaine (15 mg/kg, s.c.), reversed hyperlocomotion induced by these drugs at a dose of 0.1 μg but not at a higher dose. In the cerebral cortex homogenate, [Abu^86,94]CART (85–102)_red was nonspecifically cleaved from N- and C-termini. This peptide contains two chemically blocked Cys residues, and two others in reduced form. Concomitant with cleavage, rapid cyclization occurred. The newly formed cyclic peptides were stable. The cyclic peptide [Abu^86,94]CART (85–102)_ox failed to inhibit amphetamine- and cocaine-induced locomotor activity. The ability to inhibit the locomotor-stimulant activity of amphetamine was retained in [Abu^86,88,94,101]CART (85–102), in which all Cys were replaced with 2-aminobutyric acid to prevent their pairing. Disulfide bridge formation may be an interesting mechanism that prevents proteolysis of [Abu^86,94]CART (85–102)_red and terminates its ability to reverse amphetamine-induced hyperlocomotion.     

CART peptide: central mediator of leptin-induced adipose tissue apoptosis?

Because of connections between CART peptide containing neurons and the sympathetic nervous system (SNS) and the possible role of the SNS in leptin-induced adipose apoptosis, CART may act as a downstream effector of leptin-induced adipose apoptosis. Male Sprague-Dawley rats received continuous intracerebroventricular (i.c.v.) infusion for 4 days of either artificial cerebrospinal fluid (aCSF, 12 microl/day), leptin (15 microg/day), or CART55-102 at 2.4 microg/day (CART2.4) or 9.6 microg/day (CART9.6). Food intake (FI) was decreased 10.8% for CART2.4, 41.9% for CART9.6 and 33.4% for leptin (p<0.05). CART9.6 and leptin reduced meal size and meal number. Body weight (BW) was reduced by CART9.6 (14.6%) and leptin (11.6%) (p<0.05), but not by CART2.4. CART9.6 and CART2.4, but not leptin, caused hypothermia, and CART9.6 inhibited physical activity (p<0.05). Epididymal, inguinal and retroperitoneal fat pad weights were reduced (p<0.05) by both CART treatments and leptin; CART9.6 also reduced gastrocnemius muscle weight (18.1%, p<0.05). Leptin, but not CART, increased serum free fatty acid concentrations by 31.1% (p<0.05) and increased adipose apoptosis by 48% (p<0.05). These data show that although leptin and CART55-102 have some similar actions, CART55-102 is probably not a mediator for leptin-induced adipose apoptosis in the brain.     

CART peptide 55-102 microinjected into the nucleus accumbens inhibits the expression of behavioral sensitization by amphetamine

CART peptide has been shown to regulate the actions of psychomotor stimulants. Here we have further investigated the role of the biologically active CART 55-102 peptide in the nucleus accumbens (NAcc) in the expression of behavioral sensitization by amphetamine (AMPH). Rats were pre-exposed 5 times to either saline or AMPH (1 mg/kg, i.p.). After 2 weeks of withdrawal, rats were microinjected into the NAcc with saline or CART 55-102 (1.0, or 2.5 microg/0.5 microl/side) followed by AMPH challenge (1 mg/kg, i.p.). The enhanced increase of locomotion and rearing produced by repeated AMPH pre-exposures was dose-dependently inhibited by microinjection into the NAcc of CART 55-102 peptide. These results indicate that CART 55-102 peptide in the NAcc can play a compensatory inhibitory role in the expression of behavioral sensitization by AMPH and further suggest that CART peptide may be a useful target to control the drug addiction by psychomotor stimulants.