Received for publication July 9, 2004.
Revised December 3, 2004.
Accepted for publication December 17, 2004.

NPFF analogs functionally antagonize opioid activities in NPFF₂ receptor-transfected SH-SY5Y neuroblastoma cells

Abstract

To elucidate the mechanism of the cellular anti-opioid activity of NPFF, we have transfected the SH-SY5Y neuroblastoma cell line, which expresses µ and -opioid receptors, with the human NPFF₂ receptor. The selected clone, SH₂-D9, expressed high affinity NPFF₂ receptors in the same range order as µ and -opioid receptors (100-300 fmoles/mg protein). The NPFF analog 1DMe, did not modify the binding parameters of the µ and specific agonists, DAMGO and deltorphin-I, respectively. 1DMe dose-dependently inhibited 75-80 % of the cAMP production stimulated by forskolin. Pre-incubation 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. Pre-incubation with 1DMe reduced the maximal effect of deltorphin-I by 40 %, demonstrating an anti-opioid effect in this experimental model for the first time. By using peptides corresponding to the carboxyl-terminus of the _i1,2, _i3, _o and _s subunits of G-proteins, which specifically uncouple receptors from G-proteins we demonstrated that µ-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 G_o and G_i1,2, whereas the coupling with G_s reduced the anti-opioid 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.

Key words: Muscarinic cholinergic, Neuropeptides, Opioid, Gi family, Gs family, Gq/11 family, Calcium (G Protein Coupled Signals), Ca imaging, Receptor binding studies