Co-incident signalling between the Gi/Go-coupled d-opoid receptor and the Gq-coupled m3 muscarinic receptor at the level of intracellular free calcium in SH-SY5Y cells

In SH-SY5Y cells, activation of delta -opioid receptors with [D-Pen(2,5)]-enkephalin (DPDPE; 1 muM) 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 muM, Hill slope 1) and the concentration-response relationship for carbachol's permissive action in revealing the delta -opioid receptor-mediated elevation of [Ca2+] (EC50 0.7 mM; Hill slope 1.8). Sequestration of free G protein beta gamma dimers by transient transfection of cells with a beta gamma binding protein (residues 495-689 of the C terminal tail of G protein-coupled receptor kinase 2) reduced the ability of delta opioid receptor activation to elevate [Ca2+](i). However, DPDPE did not elevate either basal or oxotremorine-M-evoked inositol phosphate production indicating that delta -opioid receptor activation did not stimulate phospholipase C. Furthermore, delta -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 delta -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 delta -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.