Unique, Specific CART Receptor-Independent Regulatory Mechanism of CART(55-102) Peptide in Spinal Nociceptive Transmission and Its Relation to Dipeptidyl-Peptidase 4 (DDP4).

Cocaine- and amphetamine-regulated transcript (CART) peptides are involved in several physiological and pathological processes, but their mechanism of action is unrevealed due to the lack of identified receptor(s). We provided evidence for the antihyperalgesic effect of CART(55-102) by inhibiting dipeptidyl-peptidase 4 (DPP4) in astrocytes and consequently reducing neuroinflammation in the rat spinal dorsal horn in a carrageenan-evoked inflammation model. Both naturally occurring CART(55-102) and CART(62-102) peptides are present in the spinal cord. CART(55-102) is not involved in acute nociception but regulates spinal pain transmission during peripheral inflammation. While the full-length peptide with a globular motif contributes to hyperalgesia, its N-terminal inhibits this process. Although the anti-hyperalgesic effects of CART(55-102), CART(55-76), and CART(62-76) are blocked by opioid receptor antagonists in our inflammatory models, but not in neuropathic Seltzer model, none of them bind to any opioid or G-protein coupled receptors. DPP4 interacts with Toll-like receptor 4 (TLR4) signalling in spinal astrocytes and enhances the TLR4-induced expression of interleukin-6 and tumour necrosis factor alpha contributing to inflammatory pain. Depending on the state of inflammation, CART(55-102) is processed in the spinal cord, resulting in the generation of biologically active isoleucine-proline-isoleucine (IPI) tripeptide, which inhibits DPP4, leading to significantly decreased glia-derived cytokine production and hyperalgesia.

Glial cell type-specific changes in spinal dipeptidyl peptidase 4 expression and effects of its inhibitors in inflammatory and neuropatic pain.

Altered pain sensations such as hyperalgesia and allodynia are characteristic features of various pain states, and remain difficult to treat. We have shown previously that spinal application of dipeptidyl peptidase 4 (DPP4) inhibitors induces strong antihyperalgesic effect during inflammatory pain. In this study we observed low level of DPP4 mRNA in the rat spinal dorsal horn in physiological conditions, which did not change significantly either in carrageenan-induced inflammatory or partial nerve ligation-generated neuropathic states. In naïve animals, microglia and astrocytes expressed DPP4 protein with one and two orders of magnitude higher than neurons, respectively. DPP4 significantly increased in astrocytes during inflammation and in microglia in neuropathy. Intrathecal application of two DPP4 inhibitors tripeptide isoleucin-prolin-isoleucin (IPI) and the antidiabetic drug vildagliptin resulted in robust opioid-dependent antihyperalgesic effect during inflammation, and milder but significant opioid-independent antihyperalgesic action in the neuropathic model. The opioid-mediated antihyperalgesic effect of IPI was exclusively related to mu-opioid receptors, while vildagliptin affected mainly delta-receptor activity, although mu- and kappa-receptors were also involved. None of the inhibitors influenced allodynia. Our results suggest pathology and glia-type specific changes of DPP4 activity in the spinal cord, which contribute to the development and maintenance of hyperalgesia and interact with endogenous opioid systems.

The dipeptidyl peptidase IV (CD26, EC 3.4.14.5) inhibitor vildagliptin is a potent antihyperalgesic in rats by promoting endomorphin-2 generation in the spinal cord.

We have reported previously that the dipeptidyl peptidase IV inhibitor Ile-Pro-Ile had an antihyperalgesic action in rats when given intrathecally in the carrageenan-induced hyperalgesia, as detected by the Randall-Selitto test. Vildagliptin, a non-peptide inhibitor of the same enzyme, which is already on the market as an "euglycemic" agent in diabetics, has a slightly more potent and more sustained antihyperalgesic effect in the same test when given by the same route. The action of 3nmol/rat vildagliptin could be antagonized by subcutaneous naltrexone (0.5mg/kg) pretreatment, or by intrathecally co-administered specific antiserum to endomorphin-2. Thus, the antihyperalgesia by vildagliptin, similarly to Ile-Pro-Ile, was opioid receptor-mediated and could be attributed to the promotion of endomorphin-2 generation in rat spinal cord dorsal horn. Furthermore, vildagliptin (1mg/kg) is a potent antihyperalgesic also when given subcutaneously.

Projection neurons in lamina I of rat spinal cord with the neurokinin 1 receptor are selectively innervated by substance p-containing afferents and respond to noxious stimulation.

Lamina I of the spinal cord is densely innervated by nociceptive primary afferents, many of which contain substance P. It contains numerous projection neurons: the majority of these respond to noxious stimuli, however some are activated by cooling. In the rat, approximately 80% of the projection neurons express the neurokinin 1 (NK1) receptor, on which substance P acts, and most cells with this receptor are activated by noxious stimuli. Lamina I neurons can be classified morphologically into pyramidal, multipolar, and fusiform types. It has been reported in the cat that pyramidal neurons are activated only by cooling and that in monkey relatively few pyramidal cells are NK1 receptor-immunoreactive. We have used immunocytochemistry to examine the innervation of lamina I projection neurons in the rat by substance P-containing primary afferents and their responses to a noxious stimulus (subcutaneous formalin injection). NK1 receptor-immunoreactive projection cells received a significantly higher density of contacts from substance P-containing afferents than neurons that lacked the receptor. Most contacts on NK1 receptor-immunoreactive cells were associated with synapses. Formalin injection induced c-Fos in approximately 80% of projection neurons with the NK1 receptor and in 25-45% of those without it. More than 80% of pyramidal neurons expressed the receptor, and for both substance P innervation and c-Fos expression there were no significant differences among different morphological types of NK1 receptor-immunoreactive neuron. We conclude that presence or absence of the NK1 receptor is a better indicator of function than morphology for lamina I projection neurons in the rat.