JIOMICS

JIOMICS | VOL 5 | ISSUE 2 | DECEMBER 2015 | 1-62
JOURNAL OF INTEGRATED OMICS
Journal of Integrated Omics
A METHODOLOGICAL JOURNAL
HTTP://WWW.JIOMICS.COM
Special Issue: Proceeding Abstracts of the 4 th International Congress on Analytical Proteomics (ICAP 2015)
Sodium effect on agonist binding to δ-opioid receptors in isolated plasma
membranes from forebrain cortex of rats exposed to increasing doses of
morphine (10 - 50 mg/kg) for 10 days
M. Vošahlíková*, H. Ujčíková, P. Svoboda
Laboratory of Biochemistry of Membrane Receptors, Department of Biomathematics, Institute of Physiology of the Czech Academy of
Sciences, Videnska 1083, 14220 Prague 4, Czech Republic. *Corresponding author: vosahlikova@fgu.cas.cz
Available Online: 31 December 2015
Abstract
Purpose: The aim of this work was to determine the effect of sodium cations, representing the specific allosteric modulators of ligand
binding site of opioid receptors (OR), on agonist binding to δ-opioid receptors (δ-OR) in plasma membranes isolated from forebrain cortex of
rats exposed to morphine for prolonged period of time.
Experimental description: Rats were exposed to increasing doses of morphine (10 - 50 mg/kg) for 10 days and sacrificed 24 hours (group
+M10) or 20 days (group +M10/−M20) after the last dose. Control animals were sacrificed in parallel with morphine-treated (groups −M10
and (−M10/−M20)). Plasma membrane enriched fraction (PM) was isolated from rat brain cortex (+M10 and -M10) and analyzed by specific
radioligand binding. δ-OR were characterized by saturation binding studies with δ-OR agonists [ 3 H]DADLE and [ 3 H]DPDPE in the presence
or absence of 100 mM NaCl. Percoll-purified PM (from all groups) were also resolved by 1D-SDS-PAGE and 2D-ELFO. Subsequently,
immunoblot detection with specific antibodies and proteomic analysis was performed.
Results: Maximum number of [ 3 H]DADLE binding sites in PM isolated from morphine-treated rats was 1.4-fold higher than in PM isolated
from control rats. Surprisingly, 100 mM sodium chloride had no effect on [ 3 H]DADLE binding in PM prepared from morphine-treated animals
(+M10), but, as expected, it did inhibit agonist binding to PM isolated from control animals (-M10). Morphine-induced increase of ligand
binding to δ-OR was substantially higher when more specific ligand, [ 3 H]DPDPE, was used for determination of δ-OR in brain membranes.
Maximum number of [ 3 H]DPDPE binding sites in morphine-treated membranes was 2.1-fold higher than in control membranes.
Thus, the inhibition of agonist binding by sodium was detected in control membranes only. Our results indicated a specific up-regulation of
adenylyl cyclases I (8-fold) and II (2.5-fold) and significant increase of proteins functionally related to oxidative stress and apoptosis.
Conclusions: Sodium ions have been described as efficient inhibitors of agonist binding to numerous GPCR including OR, causing the
shift of receptor molecules from active (R*) to inactive (R) conformation and the uncoupling of receptor from the cognate G protein, i.e. inverse
agonist effect. The low sensitivity of δ-OR to inhibitory effect of sodium in morphine-treated samples of forebrain cortex may be interpreted
as disturbance of equilibrium between the active and non-active forms of δ-OR molecules.
Keywords: sodium, morphine, rat forebrain cortex, opioid receptors.
Acknowledgements: This work was supported by the GACR (P207/12/0919, P304/12/G069) and by the Czech Academy of Sciences (RVO:
67985823).
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