Pain and analgesia: molecular mechanisms of new therapeutic drugs and targets

Abstract

PAIN AND ANALGESIA: MOLECULAR MECHANISMS OF NEW THERAPEUTIC DRUGS AND TARGETS Yara Cury Phase1(years1-2):Characterization of the involvement of protein kinases C and MAP kinases in the antinociceptive effect of crotalphine Crotalphine is a 14 amino-acid peptide that induces potent and long-lasting antinociceptive effect mediated by activation of peripheral k- and d-opioid receptors.The intracellular mechanisms of crotalphine action involve activation of the nitric oxide-cGMP-PKG pathway with consequent opening of ATP sensitive potassium channels.Initial studies (in vitro assays using dorsal root ganglia cells)have demonstrated that crotalphine (a)induces the release of endogenous opioids, mainly dynorphin A;(b)increases the release of cGMP and (c)activates PKC z, which in turn, activates MAP kinases.These effects of crotalphine depend on k- opioid receptor activation and are potentiated by prostaglandin E2 priming.Despite these data, the involvement of PKCs and MAP kinases in the antinociceptive effect of crotalphine was not known.Moreover, a possible interaction between the oxide-cGMP-PKG pathway and PKC z- MAP kinases activation and their contribution to the long-lasting activity of the peptide were not determined yet.The aim of this study is to characterize the involvement of PKCs (isoforms µ, z, bI, bII and ´)and MAP kinases(ERK1/2, p38 e JNK)in the antinociceptive effect of crotalphine.The activities of the PD involve in vitro and in vivo studies aiming to determine:1)the effect of PKCs and MAPKs inhibitors in crotalphine antinociceptive action(behavioral studies);2)the expression of second messengers and phosphorilated kinases(in dorsal root ganglia slice obtained from animals treated with crotalphine)using Western Blotting techniques;3)the possible differences in the time course expression of second messengers, PKG, PKC and MAP kinases and their correlation with the long-lasting antinociceptive activity of the peptide.Phase2(years3-4):Characterization of cytoskeleton involvement in crotalphine antinociceptive effect The cytoskeleton is a complex and highly dynamic net of proteins that actively participates of several cellular processes,including nociceptive signaling and control. Additionally,the cytoskeleton regulates the organization and expression of membrane proteins, such as ion channels and opioid receptors, and can be modulated by the activation of these proteins.The cytoskeleton is closely involved with compartmentalization and modulation of cell signaling and can modulate the intensity or the speed of the intracellular signaling.The present study aims to characterize the involvement of cytoskeleton proteins in the antinociceptive effect of crotalphine.The activities of PD involve in vitro and in vivo studies aiming to determine:1)the effect of pharmacological disruption of cytoskeleton elements (actin microfilaments,microtubules and intermediate filaments) on crotalphine antinociceptive activity (behavioral studies);2)the effect of crotalphine in gene and protein expression of cytoskeleton accessory proteins(growth associated protein43 - Gap43, activity regulated cytoskeleton-associated protein- Arc, dynamin, clathrin heavy chain - Cltc, filamin ± - Flna, ±-tubulin, stathmin, Stop and Tau)in dorsal root ganglia;3)if the effects of crotalphine on cytoskeleton proteins is dependent on activation of opioid receptors.Phase3(year5):Characterization of cytoskeleton involvement in the release of endogenous opioids and activation of protein kinases by crotalphine Based on the results obtained in Phase2 studies, the aim of the present study is to determine the relevance of cytoskeleton elements to the intracellular signaling of crotalphine.The PD activities involve the effect of cytoskeleton disruption 1)on the release of endogenous opioid (dynorphin A,²-endorphin,met-enkephalin)by crotalphine,2)on the phosphorylation levels of protein kinases G, C and MAP kinases in dorsal root ganglion of rats treated with crotalphine