Coincident signalling between the Gi/Go-coupled δ-opioid receptor and the Gq-coupled m3 muscarinic receptor at the level of intracellular free calcium in SH-SY5Y cells

ABSTRACT

In SH-SY5Y cells, activation of δ-opioid receptors with [d-Pen^2,5]-enkephalin (DPDPE; 1 µm) did not alter the intracellular free Ca²⁺ concentration [Ca²⁺]_i. However, when DPDPE was applied during concomitant Gq-coupled m3 muscarinic receptor stimulation by carbachol or oxotremorine-M, it produced an elevation of [Ca²⁺]_i. The DPDPE-evoked increase in [Ca²⁺]_i was abolished when the carbachol-sensitive intracellular Ca²⁺ store was emptied. There was a marked difference between the concentration–response relationship for the elevation of [Ca²⁺]_i by carbachol (EC₅₀ 13 µm, Hill slope 1) and the concentration–response relationship for carbachol's permissive action in revealing the δ-opioid receptor-mediated elevation of [Ca²⁺] (EC₅₀ 0.7 mm; Hill slope 1.8). Sequestration of free G protein βγ dimers by transient transfection of cells with a βγ binding protein (residues 495–689 of the C terminal tail of G protein-coupled receptor kinase 2) reduced the ability of δ opioid receptor activation to elevate [Ca²⁺]_i. However, DPDPE did not elevate either basal or oxotremorine-M-evoked inositol phosphate production indicating that δ-opioid receptor activation did not stimulate phospholipase C. Furthermore, δ-opioid receptor activation did not result in the reversal of muscarinic receptor desensitization, membrane hyperpolarization or stimulation of sphingosine kinase. There was no coincident signalling between the δ-opioid receptor and the lysophosphatidic acid receptor which couples to elevation of [Ca²⁺]_i in SH-SY5Y cells by a PLC-independent mechanism. In SH-SY5Y cells the coincident signalling between the endogenously expressed δ-opioid and m3 muscarinic receptors appears to occur in the receptor activation-Ca²⁺ release signalling pathway at a step after the activation of phospholipase C.