Characterization of cytoskeleton involvement in crotalphine antinociceptive effect: in vivo and in vitro assays

• Grant number: 12/10105-1
• Support Opportunities: Regular Research Grants
• Start date: September 01, 2012
• End date: February 28, 2015
• Field of knowledge: Biological Sciences - Pharmacology - General Pharmacology
• Principal Investigator: Yara Cury
• Grantee: Yara Cury
• Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil

Abstract

Crotalphine, a peptide first identifies and isolated from the venom of the South American rattlesnake Crotalus durissus terrificus, displays potent and long-lasting antinociceptive activity mediated by activation of peripheral kappa- and delta-opioid receptors. Recent data have shown that crotalphine-mediated activation of opioid receptors is followed by activation of the L-arginin/NO/cGMP pathway and opening of ATP sensitive K+ channels. Experimental studies also indicated that the opioid activity of crotalphine is mainly due to the release of endogenous opioid peptides, particularly dinorphin A. In vitro studies showed that crotalphine activates MAP kinases and protein kinase Czeta. The cytoskeleton is a key element in cell signaling, and is closely involved with nociceptive and antinociceptive signaling. Cytoskeleton is a filamentous net of F-actin, microtubules and intermediate filaments, composed by one of three chemically distinct subunits, actin, tubulin or one of the several classes of intermediate filaments proteins. Additionally, a variety of accessory proteins works with the cytoskeleton and regulates its activation and consequently its effect on other proteins involved in cell signaling. Despite the importance of the cytoskeleton to antinociception, its role to the action of crotalphine was not investigated yet. Preliminary data obtained by our group, using cytoskeleton disrupting agents and a behavioral model of nociceptive evaluation, showed that actin filaments, microtubules and intermediate filaments are involved in the antinociceptive activity of crotalphine. Despite these data, it is still unclear how the interaction between crotalphine and cytoskeleton occurs and how the cytoskeleton elements can drive crotalphine action. Therefore, the aim of this work is to evaluate the role of cytoskeleton to the molecular mechanisms of crotalphine antinociceptive activity as well as the effect of the peptide in cytoskeleton structures. For this purpose, crotalphine will be administered to rats, in the presence or absence of pharmacological inhibitors of cytoskeleton structures. The effect of crotalphine will be evaluated in the model of hyperalgesia induced prostaglandin E2. The specific aims are: (1) investigate, using immunohistochemistry assays (plantar nerve and dorsal root ganglia/DRG obtained from these animals), if crotalphine interferes with cytoskeleton structures; (2) investigate, using RT-PCR and western blot assays, in DRG tissues obtained from these animals, the effect of crotalphine on the genic and proteic expression of cytoskeleton accessory proteins; (3) evaluate, using immunohistochemistry assays, the role of the cytoskeleton to the expression of mu, kappa and delta opioid receptors (plantar nerve and dorsal root ganglia/DRG); (4) investigate, by ELISA assays, the effect of pharmacological inhibitors of cytoskeleton on the release of endogenous opioids by crotalphine. (AU)