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Crotalphine: fluorescent analogues, structural analysis and its effect on co‐cultures of peritoneal macrophages and DRG neurons (LB615)
Author(s) -
Remuzgo Cesar,
Sampaio Sandra,
Picolo Gisele,
Machini Maria,
Cury Yara
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.lb615
Subject(s) - chemistry , receptor , opioid , dynorphin a , opioid peptide , substance p , hyperalgesia , microbiology and biotechnology , pharmacology , medicine , nociception , biochemistry , neuropeptide , biology
Crotalphine (CRP) is a 14‐mer peptide isolated from the venom of C. durissus terrificus “rattlesnake” that triggers long‐lasting antinociception (3‐5 days). This effect is mediated by activation of peripheral CB2 cannabinoid receptors, the subsequent release of dynorphin A and activation of opioid receptors. In inflamed tissues, granulocytes and macrophages are the main source of opioid peptides, which play a substantial role in the modulation of pain via interactions with opioid receptors located in peripheral sensory nerve fibers. In this work, we synthesized CRP and fluorescent analogues, analyzed its structure and investigated its effect on peritoneal macrophages and co‐cultures of dorsal root ganglia (DRG) neurons and peritoneal macrophages. All procedures were approved by ethics committee of the Butantan Institute (CEUAIB: 946/12) and performed in accordance to the International Association for the Study of Pain guidelines. CRP and the obtained analogues [Glu1]‐CRP, carboxyfluorescein (CF)‐[Glu1]‐CRP, CF‐[Gln1]‐CRP were able to induce antinociception. Circular dichroism spectra of CRP and [Glu1]‐CRP have shown bands indicative of the presence of random and helix structure and has allowed the construction of a 3D‐structure model of CRP. Fluorescence microscopy and mass spectra analysis of peritoneal resident or LPS‐activated macrophages treated with CF‐[Gln1]‐CRP (1 µM) showed that this peptide internalizes and is distributed in the cytoplasm of the cells, releasing dynorphin A (1‐13) and Met‐enkephalin to extracellular medium. Internalization of κ‐opioid receptors into small DRG neurons in co‐cultures of peritoneal macrophages and DRG neurons treated with bradykinin (10 μM) and CRP (1 μM), was also observed. On the other hand, we do not observed internalization of κ‐opioid receptors into macrophages. In conclusion, the data suggest a functional crosstalk between macrophages and DRG neurons in the presence of CRP. This phenomenon could contribute to the in vivo antinociceptive effect of this molecule. Grant Funding Source : Supported by FAPESP (2011/09333‐7), CeTICs/CEPID/FAPESP (2013/07467‐1) and INCTTox/CNPq (150848/2010‐2)