Journal of Neurochemistry, 2001, 76, 1688±1700 Coincident signalling between the Gi/Go-coupled d-opioid receptor and the Gq-coupled m3 muscarinic receptor at the level of intracellular free calcium in SH-SY5Y cells A. Yeo,* D. S. K. Samways,* C. E. Fowler,* F. Gunn-Moore² ,1 and G. Henderson* Departments of *Pharmacology and ²Biochemistry, University of Bristol, Bristol, UK Abstract In SH-SY5Y cells, activation of d-opioid receptors with [D-Pen 2,5 ]-enkephalin (DPDPE; 1 mM) did not alter the intracellular free Ca 21 concentration [Ca 21 ] i . However, when DPDPE was applied during concomitant Gq-coupled m3 muscarinic receptor stimulation by carbachol or oxotremorine- M, it produced an elevation of [Ca 21 ] i . The DPDPE-evoked increase in [Ca 21 ] i was abolished when the carbachol- sensitive intracellular Ca 21 store was emptied. There was a marked difference between the concentration±response relationship for the elevation of [Ca 21 ] i by carbachol (EC 50 13 mM, Hill slope 1) and the concentration±response relation- ship for carbachol's permissive action in revealing the d-opioid receptor-mediated elevation of [Ca 21 ] (EC 50 0.7 mM; Hill slope 1.8). Sequestration of free G protein bg dimers by transient transfection of cells with a bg binding protein (residues 495± 689 of the C terminal tail of G protein-coupled receptor kinase 2) reduced the ability of d opioid receptor activation to elevate [Ca 21 ] i . However, DPDPE did not elevate either basal or oxotremorine-M-evoked inositol phosphate production indicat- ing that d-opioid receptor activation did not stimulate phospholipase C. Furthermore, d-opioid receptor activation did not result in the reversal of muscarinic receptor desensi- tization, membrane hyperpolarization or stimulation of sphin- gosine kinase. There was no coincident signalling between the d-opioid receptor and the lysophosphatidic acid receptor which couples to elevation of [Ca 21 ] i in SH-SY5Y cells by a PLC-independent mechanism. In SH-SY5Y cells the coinci- dent signalling between the endogenously expressed d-opioid and m3 muscarinic receptors appears to occur in the receptor activation-Ca 21 release signalling pathway at a step after the activation of phospholipase C. Keywords: Ca 21 mobilization, d-opioid receptors, m3 muscarinic receptors, phospholipase C, receptor cross-talk, SH-SY5Y cells. J. Neurochem. (2001) 76, 1688±1700. G protein-coupled receptor activation results in the modu- lation of a number of cell signalling pathways such as adenylyl cyclase, phospholipase C (PLC) and ion channels (Neer 1995). In addition to these well documented, linear signalling pathways there is now considerable evidence for more complex coincident signalling, or cross-talk, between G protein-coupled receptors and between G protein-coupled receptors and other types of receptor (Neer 1995; Selbie and Hill 1998). In a number of cell types including neurones and neuronal cell lines, astrocytes, smooth muscle and mammalian expression systems, Gi/Go-coupled receptor activation alone does not activate the PLC/IP 3 /intracellular Ca 21 release pathway. However, in the presence of concomitant Gq coupled receptor activation a Gi/Go-coupled receptor- mediated response was revealed (Gerwins and Fredholm 1992; Okajima et al. 1993; Dickenson and Hill 1994; Connor and Henderson 1996; Connor et al. 1996, 1997a, 1997b; Biber et al. 1997; Tomura et al. 1997; Toms and Roberts 1999; Cho et al. 2000). This Gi/Go-coupled 1688 q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1688±1700 Received July 10, 2000; revised manuscript received November 3, 2000; accepted November 8, 2000. Address correspondence and reprint requests to G. Henderson, Department of Pharmacology, Medical Sciences Building, University of Bristol, University Walk, Bristol, BS8 1TD, UK. E-mail: graeme.henderson@bris.ac.uk 1 Present address: Department of Preclinical Veterinary Sciences, Royal (Dick) College of Veterinary Studies, University of Edinburgh, Summerhall, Edinburgh EH9 1QH, UK. Abbreviations used: BSA, bovine serum albumin; carbachol, carbamylcholine chloride; CHO-d, Chinese hamster ovary cells transfected with the mouse d-opioid receptor; DHS, dl-threo-dihydro- sphingosine; DPDPE, [d-Pen 2,5 ]-enkephalin; PCL, phospholipase C; GRK, G protein-coupled receptor kinase; GFP, green ¯uorescent protein; IP 1 , inositol-1-phosphate; [Ca 21 ] i , intracellular calcium; LPA, lysophosphatidic acid; oxo-M, oxotremorine-M; SEM, standard error of the mean; UTP, uridine triphosphate.