Clonación y caracterización de precursores biosinteticos de secuencias peptídicas tipo endomorfinas

Abstract

ABSTRACT: Endomorphin-1 (EM-1) and endomorphin-2 (EM-2) are the latest opioid peptide ligands discovered in the CNS of mammals that display the highest affinity and selectivity for the mu opiate receptor. EM1-2 display several, pharmacological physiological, behavioral cellular and molecular bioactivities shared by opiate alkaloids and its immunoreactivity shown to be distributed in spinal and supraspinal areas of the rat CNS that mediate pain transmission and analgesia and co-localized with MOR. . These findings support the endogenous role of these amide tetrapeptides as active neuromodulators in CNS of mammals. However, no data have been reported regarding the identification and characterization of peptide precursor(s) coding these two molecules as shown for other endogenous opioid substances. With the aim to search for the existence of such putative precursor(s) protein(s), two-antigen affinity purified antisera (AAPA) were generated against synthetic EM-1 and EM-2 peptides and their specificity and selectivity validated by a fmol sensitive-solid-phase RIA (SP-RIA) assays, respectively. Westernblot and gel permeation chromatography (coupled to SP-RIA for EM-1) assays were used to detect EM1-2-immunoreactivity (IR) in neuronal and non-neuronal tissues of the rat, including superfusates from depolarized rat striatum with 50 mM KCl. Distribution and cellular expression of EM1-2-IR were assessed by immunohistochemiscal mapping of representative rostro-caudal areas of the rat CNS. Our results show that both A-APA for EM-1 (C-14) and EM-2 (C-16) were highly specific when reacting against synthetic EM1-2 peptides and capable to detect EM1-2-IR in high and low molecular mass protein/peptide components (> 15 kDa, < 77 kDa) in rat neural tissues as revealed in western blot and chromatography assays. Chromatography profile of eluted superfusates samples revealed the presence of two-IR-peptide peaks of low-molecular weght (≈ 0.7-0.6 kDa) that cross-matched with synthetic EM-1 in RP-HPLC. Both C-14/C-16 AAPA showed the wide distribution of EM-IR in extensive neuropil, varicose and fine-axon fibers, round and oval-shaped somata of different neurons within the rat CNS. These studies provide the first immunochemical evidence of the biosynthetic origin of endomorphin molecules from putative non-identified specific propeptide precursor(s) in neurons; showing the great versality and applicability of both EM1-2 antisera in different immunochemical techniques