Neuropeptide FF (NPFF) analogs functionally antagonize opioid activities in NPFF2 receptor-transfected SH-SY5Y neuroblastoma cells

Authors:
Mollereau C, Mazarguil H, Zajac JM and Roumy M
In:
Source: Mol Pharmacol
Publication Date: (2005)
Issue: 67(3): 965-975
Research Area:
Neurobiology
Cells used in publication:
SH-SY5Y
Species: human
Tissue Origin: brain
Platform:
Nucleofectorâ„¢ I/II/2b
Experiment
Using the Nucleofector, peptides corresponding to the C-terminus of several G protein subunits were transferred into the clone SH2-D9, a derivative of the SH-SY5Y neuroblastoma cell line expressing the NPFF2 receptor. Peptide delivery efficiency was 77% as measured with an Alexa-labeled peptide.
Abstract
To elucidate the mechanism of the cellular antiopioid activity of neuropeptide FF (NPFF), we have transfected the SH-SY5Y neuroblastoma cell line, which expresses mu-and delta-opioid receptors, with the human NPFF2receptor. The selected clone, SH2-D9, expressed high-affinity NPFF2 receptors in the same range order as mu- and delta-opioid receptors (100-300 fmol/mg of protein). The NPFF analog [D-Tyr1, (NMe)Phe3]NPFF (1DMe) did not modify the binding parameters of the mu- and delta-specific agonists [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin and deltorphin-I, respectively. 1DMe dose dependently inhibited 75 to 80% of the cAMP production stimulated by forskolin. Preincubation with 1DMe halved the maximal inhibition of N-type Ca2+ channels by opioid agonists. In the presence of carbachol, acting on muscarinic receptors to release Ca2+ from the intracellular stores, deltorphin-I and 1DMe enhanced this release. Preincubation with 1DMe reduced the maximal effect of deltorphin-I by 40%, demonstrating an antiopioid effect in this experimental model for the first time. By using peptides corresponding to the carboxyl terminus of the alphai1,2, alphai3, alphao, and alphas subunits of G proteins, which specifically uncouple receptors from G proteins, we demonstrated that mu-opioid and NPFF2 receptors couple to the four subunits assayed. The Ca2+ release from the intracellular stores by 1DMe resulted from the coupling of NPFF2 receptors with Galphao and Galphai1,2, whereas the coupling with Galphas reduced the antiopioid effect of 1DMe in the modulation of N-type channels. This SH2-D9 cell line now provides the opportunity to study the interaction between both receptors.