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-Pen2,5]-enkephalin (DPDPE; 1 mM) did not alter the

intracellular free Ca21 concentration [Ca21]i. However, when

DPDPE was applied during concomitant Gq-coupled m3

muscarinic receptor stimulation by carbachol or oxotremorine-

M, it produced an elevation of [Ca21]i. The DPDPE-evoked

increase in [Ca21]i was abolished when the carbachol-

sensitive intracellular Ca21 store was emptied. There was a

marked difference between the concentration±response

relationship for the elevation of [Ca21]i by carbachol (EC50

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 [Ca21] (EC50 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

[Ca21]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 [Ca21]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-Ca21 release signalling pathway at a step after the

activation of phospholipase C.

Keywords: Ca21 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/IP3/intracellular Ca21 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.uk1Present 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-Pen2,5]-enkephalin; PCL, phospholipase C;

GRK, G protein-coupled receptor kinase; GFP, green ¯uorescent

protein; IP1, inositol-1-phosphate; [Ca21]i, intracellular calcium; LPA,

lysophosphatidic acid; oxo-M, oxotremorine-M; SEM, standard error of

the mean; UTP, uridine triphosphate.

receptor response appeared to be mediated by free G protein

bg dimers because it was reduced under conditions where

free bg dimers were sequestered by over expressed bg

binding proteins such as the bg binding domain of the G

protein-coupled receptor kinase 2 (GRK2) or the a subunit

of transducin (Chan et al. 2000; Selbie et al. 1997). A

number of different modes of action for free bg dimers

in receptor cross-talk have been proposed. These include

excess bg dimers associating with free aq dimers to

enhance the turnover of Gq (Quitterer and Lohse 1999),

synergistic activation of PLCb isozymes by aq and bg

dimers (Zhu and Birnbaumer 1996; Selbie and Hill 1998) or

sensitization of the IP3 receptor to IP3 (Neylon et al. 1998;

Xu et al. 1996; Zeng et al. 1996).

We have previously demonstrated that activation of

several types of Gi/Go-coupled receptors (d-opioid, m-opioid,

ORL1, sst2 and NPY Y2 receptors) in the SH-SY5Y human

neuroblastoma cell line required concomitant m3 muscarinic,

Gq-coupled receptor activation to reveal the Gi/Go-coupled

receptor-mediated elevation of intracellular calcium ([Ca21]i)

(Connor and Henderson 1996; Connor et al. 1996, 1997a,

1997b). The Gi/Go-coupled receptor-mediated response was

abolished by pretreatment with pertussis toxin and by deple-

tion of the [Ca21]i stores with thapsigargin. The requirement

for concomitant Gq-coupled receptor activation was absolute

in that if the Gi/Go-coupled receptor agonist was applied

simultaneously with a muscarinic receptor antagonist then

the Gi/Go-coupled receptor-mediated response was also

abolished.

In the present paper we have further characterized the

nature of the coincident signalling between the endogen-

ously expressed d-opioid and m3 muscarinic receptors. We

have sought to determine (i) if the d-opioid and m3

muscarinic receptors release Ca21 from the same intracel-

lular Ca21 store; (ii) if activation of the d-opioid receptor

reverses m3 muscarinic receptor desensitization; (iii) the

role of bg dimers from Gi/Go in the d-opioid receptor

elevation of [Ca21]I; (iv) if the coincident signalling is

mediated through concomitant activation of PLCb; and (v) if

the d-opioid receptor exhibits cross-talk with a receptor

which elevates [Ca21]i through a PLC-independent path-

way. Some of this work has appeared previously in abstract

form (Yeo et al. 1996; Yeo and Henderson 1997a,1997b).

Materials and methods

Cell culture

SH-SY5Y cells were cultured as described previously (Connor and

Henderson 1996). Chinese hamster ovary cells transfected with the

mouse d-opioid receptor (CHO-d) were cultured in Nutrient Mixture

Ham's F12 Medium supplemented with penicillin (100 i.u./mL),

streptomycin (100 mg/mL), amphotericin B (2.5 mg/mL), geneticin

(100 mg/mL) and fetal bovine serum (10%) in a humidi®ed

incubator with 5% CO2. For the monolayer studies, cells were

plated on to plastic slides and cultured in Leighton tubes (Costar),

whereas for single cell imaging experiments cells were plated at a

low density on to glass coverslips. For measurement of inositol

phosphate production, 4 � 12-well plates were seeded with cells

and the experiments performed once the cells had reached

con¯uence.

Cell transfection

SH-SY5Y cells were transiently cotransfected with a mutant green

¯uorescent protein (S65T GFP) as a marker for successful

transfection and a G protein bg dimer binding protein comprising

part of the C terminal region (amino acids 495±689) of G protein-

coupled receptor kinase 2 (GRK2). Cells were transfected at

378C by incubation for 2 h with a mixture of Tfx-50 (Promega)

and the plasmids containing the construct for S65T GFP (pCMV-

GFP) and the construct for the bg dimer binding protein

(pcDNAneo-GRK2495±689) at a Tfx : DNA ratio of 3 : 1 and a

pCMV-GFP : pcDNAneo-GRK2495±689 ratio of 1 : 4. 24 h after

transfection this protocol resulted in 30±50% of cells exhibiting

GFP ¯uorescence observed using a 490-nm excitation ®lter and a

520-nm broad-pass emission ®lter. GFP-positive cells were assumed

to be coexpressing the G protein bg dimer binding protein.

Measurement of intracellular Ca21

Intracellular free Ca21 concentration ([Ca21]i) was measured using

the ¯uorescent Ca21-sensitive dye fura-2 as described previously

(Connor and Henderson 1996). Brie¯y, cells were superfused with

a buffer containing: NaCl 140 mM, KCl 2 mM, CaCl2 2.5 mM,

MgCl2 1 mM, HEPES 10 mM, glucose 10 mM, sucrose 40 mM,

bovine serum albumin (BSA) 0.05%, pH 7.3. In those experiments

in which lysophosphatidic acid was studied, fatty acid free BSA

was used.

Monolayers

After loading fura-2 the plastic slides were ®xed in a quartz cuvette.

The cuvette was then placed in an LS-5B Perkin-Elmer spectro-

¯uorimeter and perfused with buffer (4 mL/min at 378C). Drugs

were added to the perfusion buffer in known concentrations. The

spectro¯uorimeter was controlled by a computer running a Perkin-

Elmer software package. The fura-2 loaded cells were alternately

exposed to light at 340 nm and 380 nm and the emission of the

cells at 510 nm was recorded.

Single cell imaging

After loading fura-2, the coverslips were transferred into a 1-mL

chamber (maintained at 378C), placed onto an inverted microscope

(Nikon Diaphot) and superfused with buffer at 4 mL/min. Drugs

were added to the perfusion buffer in known concentrations. Cells

were initially categorized as GFP-positive or GFP-negative (see

above) and then alternately exposed to light at 340 nm and 380 nm

and the intensity of the emission of the cells at 510 nm was

measured using a Photonic Science camera. Ionvision software was

used to collect data and also to control the position of the ®lter

wheel selecting the excitation wavelength. Fluorescence intensity

was measured at the two emission wavelengths every 5 s or every

20 s (acquisition speed being faster during drug application and

slower during periods of washout). Background ¯uorescence

intensity was taken from an area of the coverslip with no cells.

Groups of 5±20 cells were imaged in any single experiment.

Co-incident signalling between d-opioid and m3 muscarinic receptors 1689

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1688±1700

Measurement of [3H] inositol phosphate accumulation

Phosphoinositide hydrolysis was determined by monitoring the

accumulation of inositol monophosphate in the presence of LiCl as

described previously (Toms et al. 1995). SH-SY5Y and CHO-d

cells were incubated with 4 mCi/mL [3H]inositol for 20±24 h at

378C to allow incorporation of [3H] into inositol phosphates.

Prelabelled cells were washed with a buffer containing: NaCl

118 mM, KCl 4.7 mM, NaHCO3 25 mM, KHPO4 1.2 mM, CaCl21.2 mM, MgSO4 1.2 mM, HEPES 10 mM, glucose 10 mM and

then incubated with Li1 (10 mm) for 20 min. Drugs were then

added as required, the reaction was terminated by aspiration and

addition of 500 mL ice cold perchloric acid (7.5%). Total inositol

phosphates were extracted by freeze thawing, and IP1 separated

using Dowex chromatography. The amount of [3H]-IP1 was

determined by scintillation counting.

Whole-cell patch clamp

Whole-cell patch clamp experiments were performed at 19±218C

as described previously (Seward and Henderson 1990). The super-

fusing solution consisted of: NaCl 140 mM, KCl 2 mM, CaCl22.5 mM, MgCl2 1 mM, HEPES 10 mM, glucose 10 mM, sucrose

40 mM, pH 7.4; and the pipette solution: KCl 140 mM, MgCl21 mM, CaCl2 1 mM, EGTA 10 mM, Na2 ATP 5 mM, NaGTP

0.2 mM, HEPES 10 mM, pH 7.2. The cells were superfused at

2.5 mL/min. Pipette resistances were 2±5 mV.

Drugs and chemicals

N-acetylsphingosine, apyrase, atropine methylbromide, carbamyl-

choline chloride (carbachol), dl-threo-dihydrosphingosine (DHS),

[d-Pen2,5]-enkephalin (DPDPE), fura-2AM, lysophosphatidic acid

(LPA), mecamylamine hydrochloride, oxotremorine-M (oxo-M)

and uridine triphosphate (UTP) were obtained from (Sigma).

[3H] inositol was obtained from American Radiolabelled Chemicals

Ltd.

Data analysis

Data are reported as mean ^ standard error of the mean (SEM);

statistical comparisons were made using either an unpaired

Student's t-test or a Chi-square test as appropriate; p , 0.05 was

considered signi®cant.

Results

Coincident signalling between d-opioid receptors and

m3 muscarinic receptors

When applied to SH-SY5Y cells the d-opioid receptor

agonist DPDPE on its own did not elevate [Ca21]i whereas

either carbachol (1 mm, Fig. 1a) or oxo-M (1 mm), acting at

Gq-coupled muscarinic receptors in these cells, evoked a

biphasic elevation of [Ca21]i. However, in the continued

presence of carbachol or oxo-M, application of DPDPE now

evoked a rapid elevation of [Ca21]i (Fig. 1a).

It was not possible with population studies on cell

monolayers to determine whether the same cells were

responding to muscarinic and d-opioid receptor activation.

We therefore employed single cell imaging of [Ca21]i to

identify the cells that responded to Gq-and Gi/Go-coupled

receptor activation. 95% of cells studied responded to

oxo-M (1 mm) with a rise in [Ca21]i (Table 1a). In the

absence of oxo-M a few cells (5%) responded to DPDPE

(1 mm) alone but there was no correlation between a lack of

response to oxo-M and a response to DPDPE in the absence

of oxo-M. In the continued presence of oxo-M (1 mm) 60%

of the oxo-M-responding cells exhibited an elevation of

[Ca21]i in response to DPDPE (1 mm).

We next sought to compare the concentration-dependence

of the carbachol elevation of [Ca21]i with that for its

permissive role in revealing the d-opioid receptor-mediated

elevation of [Ca21]i. In monolayers carbachol alone evoked

a concentration-dependent elevation of [Ca21]i (Figs 1b

and c). The EC50 for carbachol was 13 mm (95% con®dence

limits 1±137 mm), the Hill slope was not signi®cantly

different from unity and the response reached a maximum at

1 mm. In these experiments mecamylamine (100 mm) was

present to exclude any contribution at high agonist con-

centrations of nicotinic receptors present in SH-SY5Y cells

(Forsythe et al. 1992). The maximum response to carbachol

(3 mm) in the presence of mecamylamine was 93 ^ 14%

(n � 3) of the response seen to a maximally effective

concentration of the selective muscarinic receptor agonist,

oxo-M (30 mm). We then determined the concentration-

dependence of the permissive action of carbachol to reveal

the d-opioid receptor-mediated elevation of [Ca21]i by

applying a ®xed concentration of DPDPE (30 nm) in the

presence of increasing concentrations of carbachol (Figs 1b

and c). The amplitude of the response evoked by DPDPE

(30 nm) increased as the concentration of carbachol was

increased, reaching 50% of its maximum in the presence of

720 nm carbachol (95% con®dence limits 305 nm to

1.7 mm). The concentration±response curve for carbachol

had a Hill slope of 1.8 and was maximal at 10 mm carbachol.

Thus the concentration±response curve for carbachol's

permissive action to reveal the DPDPE elevation of

[Ca21]i lies approximately 18-fold to the left of that for

carbachol alone elevating [Ca21]i.

d-Opioid receptor activation releases Ca21 from the

same intracellular stores as m3 muscarinic receptor

activation

To determine whether d-opioid receptor activation released

Ca21 from the same intracellular stores as Gq-coupled

muscarinic receptor activation we sought to empty the

muscarinic receptor-sensitive stores by exposing the cells to

a maximally effective concentration of carbachol in the

absence of extracellular Ca21. In the absence of extra-

cellular Ca21 the carbachol-sensitive Ca21 stores should be

prevented from re®lling through capacitative Ca21 entry

pathways (Grudt et al. 1996). When carbachol (1 mm) was

applied in the absence of extracellular Ca21 it evoked a

large transient rise in [Ca21]i that returned to baseline levels

even in the continued presence of the drug (Fig. 2).

Following exposure to high carbachol, the cells failed to

1690 A. Yeo et al.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1688±1700

respond when challenged with another Gq-coupled receptor

agonist, bradykinin (1 mm) that prior to carbachol evoked a

rise in [Ca21]i, indicating that indeed the Ca21 stores had

been emptied (data not shown). In the absence of extra-

cellular Ca21 but in the presence of a low concentration of

carbachol (1 mm) (i.e. when the intracellular Ca21 stores had

not been depleted) DPDPE (30 nm) evoked an elevation in

[Ca21]i (Fig. 2a). However, when applied in the presence of

the high carbachol concentration (1 mm), in the absence of

extracellular Ca21 (i.e. when the intracellular Ca21 stores

were depleted), DPDPE (30 nm) failed to evoke a rise in

[Ca21]i (Fig. 2b). These data demonstrate that the d-opioid

receptor-mediated rise in [Ca21]i resulted from Ca21 release

from the same stores as the Gq-coupled muscarinic receptor.

d-Opioid receptor activation does not reverse m3

muscarinic receptor desensitization

One possible explanation for the coincident nature of the

d-opioid receptor and muscarinic receptor elevations of

[Ca21]i is that stimulation of the d-opioid receptors in some

way reversed the desensitization of muscarinic receptors

developing during the continuous exposure to the muscarinic

agonist. If this was the case then it would be expected that

the DPDPE-evoked rise in [Ca21]i would be smallest if

applied at the start of the exposure to carbachol and be larger

if applied at a later time point as the desensitization of the

muscarinic receptors increased. To investigate this, DPDPE

(30 nm) was applied in the continued presence of carbachol

(1 mm) at various time points (0, 1, 2 and 10 min) after the

Fig. 1 Carbachol concentration±response

relationships. (a) The trace represents a

continuous record of [Ca21]i in a monolayer

of SH-SY5Y cells. DPDPE alone did not

elevate [Ca21]i whereas carbachol pro-

duced a biphasic elevation of [Ca21]i. In the

presence of carbachol, DPDPE now also

elevated [Ca21]i. Drugs were applied for the

periods indicated by the bars. (b) The two

traces represent continuous records of

[Ca21]i in monolayers of SHSY5Y cells

taken from two halves of the same cover-

slip. Carbachol produces a concentration-

dependent elevation of [Ca21]i. At the

low carbachol concentration (300 nM) the

response to DPDPE (30 nM) was small,

whereas in a higher carbachol concentra-

tion (3 mM) the response evoked by DPDPE

(30 nM) was larger. (c) The concentration-

response curve for the peak elevation of

[Ca21]i by carbachol lies to the right of that

for carbachol revealing the DPDPE-evoked

elevation of [Ca21]i. The closed circles

represent the concentration±response

curve for the peak elevation of [Ca21]i by

carbachol. The experiment was performed

in the presence of the nicotinic antagonist

mecamylamine (100 mM). The open circles

represent the concentration-response curve

for carbachol revealing the DPDPE evoked

elevation of [Ca21]i. Monolayers of cells

were exposed to a ®xed concentration of

DPDPE (30 nM) in the presence of different

concentrations of carbachol. Each point

represents the mean data obtained from

between 5 and 9 experiments, the bars

represent the SEM.

Co-incident signalling between d-opioid and m3 muscarinic receptors 1691

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1688±1700

beginning of the carbachol application (Fig. 3). There was

no signi®cant difference in the responses evoked at the

different time points. The mechanism by which DPDPE

elevated [Ca21]i in the continued presence of carbachol

therefore does not appear to be reversal of muscarinic

receptor desensitization.

Role of G protein bg dimers in d-opioid

receptor-mediated elevation of [Ca21]i

G protein-coupled receptor kinases (GRK) 2 and 3 bind to

bg dimers using a plekstrin homology domain contained in

their C terminal regions (Koch et al. 1993). This interaction

normally facilitates the translocation of GRKs to the plasma

membrane. Use can also be made of this interaction to

examine the role of bg dimers in cellular processes because

in cells overexpressing the C terminal portion of GRK 2 or 3

this binding protein will sequester bg dimers and thus

inactivate them (Inglese et al. 1994).

SH-SY5Y cells were transiently cotransfected with two

plasmids, one containing an insert coding for GFP and the

other an insert encoding for the C terminal region (amino

acids 495±689) of GRK2. Successfully transfected cells

were identi®ed by their green ¯uorescence. Because only a

proportion of cells (30±50%) were successfully transfected

we used single cell imaging to determine if transfection with

a bg binding protein decreased the response to m3

muscarinic and d-opioid receptor activation. Even in non-

transfected cells there was considerable variability in the

amplitude of the responses to m3 muscarinic and d-opioid

receptor activation in different cells. Therefore in these

experiments we considered a cell to be responsive if the

application of an agonist raised [Ca21]i above basal levels.

In the population of successfully transfected cells the

number of cells responding to oxo-M (1 mm) with an

increase in [Ca21]i was not different from cells transfected

with the vector alone or from wild-type cells (Table 1b).

However, the proportion of cells responding to DPDPE

(1 mm) was signi®cantly lower in cells transfected with the

bg binding protein (Chi-square test, p , 0.05). These data

indicate that the d-opioid receptor-mediated elevation of

[Ca21]i was mediated through bg dimers.

Potential role of PLCb activation in d-opioid

receptor-mediated elevation of [Ca21]i

To examine whether Gi/Go-coupled receptor activation

altered PLCb activity we measured inositol-1-phosphate

Table 1 (a) Number of SH-SY5Y cells exhibiting an elevation of [Ca21]i in response to oxo-M, DPDPE and DPDPE in the presence of oxo-M

Drug treatment Number of cells responding

Oxo-M (1 mM) 112 of 118 (95%)

DPDPE (1 mM) 6 of 118 (5%)

DPDPE (1 mM) in the presence of oxo-M (1 mM) 67 of 112 (60%)

Data are from eight coverslips on each of which 11±23 cells were examined.

(b) Effect of transient transfection of SH-SY5Y cells with a bg binding protein on the elevation of [Ca21]i by oxo-M and DPDPE in the presence of

oxo-M

Number of cells responding

Non-transfected (GFP-negative) Transfected (GFP-positive)

Transfection Oxo-M (1 mM)

DPDPE (1 mM)

in the presence of oxo-M (1 mM) Oxo-M (1 mM)

DPDPE (1 mM)

in the presence of oxo-M (1 mM)

bg binding protein 216 of 236 145 of 216 36 of 43 10 of 36*

(92%) (67%) (84%) (28%)

[0.950] [0�.278] [0�.650]1 [0.000]11

Vector control 58 of 67 28 of 58 24 of 28 16 of 24

(87%) (48%) (86%) (66%)

Cells were transiently transfected with a mixture of two plasmids; one containing the insert for GFP and one containing either the insert for amino

acids 495±689 of GRK2 (bg binding protein) or no insert as control (vector control). Cells expressing GFP (30±50% of total) were assumed to also

be expressing the bg binding protein. For each drug treatment the data were obtained from 8±10 coverslips on each of which a number of cells

were examined. *Indicates a signi®cant difference (p , 0.05, Chi-square test) compared with non-transfected cells and with cells transfected with

the vector alone. Values in square brackets are the median increases in the ratio of ¯uorescence emission at 340 : 380 nm. 1 and11 indicate

signi®cant difference from non-transfected cells at the 0.01 and 0.001 levels, respectively, determined using a Mann±Whitney U-test.

1692 A. Yeo et al.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1688±1700

(IP1) accumulation in the presence of Li1. Oxo-M (0.1±

100 mm) evoked a concentration-dependent stimulation of

IP1 accumulation (Fig. 4a). The concentration±response

curve for oxo-M stimulation of IP1 production lies slightly

to the left of that for oxo-M elevation of [Ca21]i, with both

curves having Hill slopes close to unity (Fig. 4a). We then

used a concentration of oxo-M (1 mm) that evoked a similar

rise in [Ca21]i as did a maximally effective concentration of

DPDPE (1 mm). DPDPE did not alter either the basal level

of IP1 production or the increase in IP1 production evoked

by oxo-M at 2, 5 or 20 min (Fig. 4b).

It was surprising that in SH-SY5Y cells the coincident

signalling between d-opioid receptors muscarinic receptors

did not result in enhanced PLC activation and thus an

increase in IP1 production given that other workers, using

other cell types have reported an interaction at the level of

PLC (Gerwins and Fredholm 1992; Okajima et al. 1993;

Dickenson and Hill 1994; Biber et al. 1997; Tomura et al.

1997; Selbie and Hill 1998; Chan et al. 2000). To ensure

that the lack of PLC activation in SH-SY5Y cells was not

some methodological artefact we examined whether we

could observe d-opioid receptor activation of PLC in another

cell type. In monolayers of CHO cells stably transfected

with the d-opioid receptor (CHO-d cells), DPDPE (30±

1000 nm) applied alone did not elevate [Ca21]i (Fig. 5a).

However, in the presence of UTP (1 mm), an agonist at Gq

Fig. 2 Depletion of the carbachol-releasa-

ble Ca21 pool prevented the DPDPE ele-

vation of [Ca21]i. The traces represent

continuous records of [Ca21]i in monolayers

of SH-SY5Y cells. Drugs were applied for

the periods indicated by the bars. (a) In the

absence of extracellular Ca21 both carba-

chol (1 mM) and DPDPE (in the continued

presence of carbachol) evoked a transient

increase in [Ca21]i. (b) In the absence of

extracellular Ca21 a maximally effective

concentration of carbachol (1 mM) caused a

large transient rise in [Ca21]i that declined

back to baseline levels in the continued

presence of carbachol. Thereafter, in the

continuous presence of carbachol, DPDPE

(30 nM) failed to elevate [Ca21]i. The insert

shows an enlarged portion of the trace

during the application of DPDPE.

Co-incident signalling between d-opioid and m3 muscarinic receptors 1693

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1688±1700

coupled P2Y receptors in these cells which evoked a

biphasic increase in [Ca21]i, DPDPE now caused a rapid

elevation in [Ca21]i. We then measured IP1 accumulation in

the presence of Li1 in CHO-d cells. UTP (1±1000 mm)

evoked a concentration-dependent elevation of IP1 accumu-

lation in CHO-d cells (Fig. 5b). In contrast to SH-SY5Y

cells, in CHO-d cells the concentration-response curve for

agonist stimulation of IP1 production lies slightly to the right

of that for agonist elevation of [Ca21]i, both curves having

Hill slopes close to unity (Fig. 5b).We used a concentration

of UTP (10 mm) which evoked a similar rise in [Ca21]i as a

maximally effective concentration of DPDPE (1 mm).

DPDPE (1 mm) potentiated the UTP-evoked accumulation

of IP1 at 2, 5 and 20 min but did not affect the basal

accumulation of IP1 at the same time points (Fig. 5c). Thus,

in the presence of concomitant Gq coupled receptor activa-

tion d receptor activation stimulates PLC in CHO-d cells but

not SH-SY5Y cells.

Sphingosine kinase does not mediate the d-opioid

receptor-mediated elevation of [Ca21]i

Activation of sphingosine kinase leads to the generation

of sphingosine-1-phosphate which is an alternative second

messenger for intracellular Ca21 mobilization (Beaven

Fig. 3 The amplitude of the DPDPE evoked elevation of [Ca21]i

was independent of the time after the start of the carbachol applica-

tion at which DPDPE was applied. (a) Three superimposed traces of

[Ca21]i from the same coverslip of SH-SY5Y cells. These traces

show (i) a control response to carbachol (1 mM), (ii) a response to

DPDPE (30 nM) applied 1 min after the beginning of the carbachol

exposure, and (iii) a response to DPDPE (30 nM) applied 10 min

after the beginning of the carbachol exposure. To overlay the traces

the ratios of 340/380 nm have been normalized to 1 at the start of

each trace. (b) Comparison of the rise in [Ca21]i evoked by DPDPE

(30 nM) applied at 0, 1, 2 and 10 mins after the beginning of the

carbachol exposure. Histograms represent the mean and SEM from

4 to 7 experiments and each response has been normalized to the

amplitude of the response to DPDPE applied 10 min after the start

of the carbachol exposure.

Fig. 4 Effect of oxo-M and DPDPE on IP1 production. (a) Concen-

tration±response relationships for oxo-M-evoked elevation of [Ca21]i

(B) and accumulation of IP1 (A) in SH-SY5Y cells. The data repre-

sent the mean ^SEM from 4 experiments. (b) Time course of IP1

accumulation in SH-SY5Y cells under the following conditions (i)

basal (A), (ii) in the presence of DPDPE (1 mM)(B), (iii) in the

presence of oxo-M (1 mM) (K), and (iv) in the presence of oxo-M

(1 mM) plus DPDPE (1 mM)(O). Each point represents the mean

^SEM of 3±7 experiments.

1694 A. Yeo et al.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1688±1700

1996). Inhibitors of sphingosine kinase have been shown to

block m2 muscarinic receptor-mediated elevation of [Ca21]i

(Meyer zu Heringdorf et al. 1998). The sphingosine kinase

inhibitor, dl-threo-dihydrosphingosine (DHS, 100 mm), was

applied to SHSY5Y cells for 10 min prior to and during

agonist application. DHS did not change the amplitude of

the response to oxo-M (1 mm) and augmented, rather than

inhibited, the response to DPDPE (1 mm). After DHS

treatment the responses to oxo-M were 111 ^ 31% of con-

trol (n � 3, p . 0.05) whereas those to DPDPE were

198 ^ 44% of control (n � 3, p , 0.05). The inactive

analogue of DHS, N-acetylsphingosine (100 mm) which

does not inhibit sphingosine kinase at micromolar concen-

trations (Yatomi et al. 1996) had no effect on the responses

to oxo-M or DPDPE (data not shown).

We next sought to determine if the d-opioid receptor

exhibited cross-talk with a G protein-coupled receptor

whose activation gave rise to an elevation of [Ca21]i

independent of PLC activation and inositol phosphate

production. One such PLC-independent pathway is through

sphingosine kinase activation (Meyer zu Heringdorf et al.

1998). In SH-SY5Y cells the elevation of [Ca21]i by

lysophosphatidic acid (LPA) was independent of PLC and

was blocked by a sphingosine kinase inhibitor (Young et al.

1999). In monolayers of SH-SY5Y cells LPA (30 nm-

10 mm) evoked a transient increase in [Ca21]i that declined

to basal levels or just above basal levels (see Fig. 6a) in the

continued presence of LPA. In the continued presence of

LPA, coapplication of DPDPE did not evoke a further

elevation of [Ca21]i (Fig. 6a). Because the sustained plateau

elevation of [Ca21]i by LPA was absent in some experi-

ments and in the other experiments was much less than that

evoked by carbachol we also sought to determine if d-opioid

receptor activation could elevate the initial peak response to

LPA. When DPDPE (1 mm) was co-applied with a sub-

maximal concentration of LPA (1 mm) the amplitude of the

peak elevation of [Ca21]i was not different from that evoked

by LPA alone (Fig. 6b). For comparison, when DPDPE was

coapplied with carbachol (1 mm) the amplitude of the

elevation of [Ca21]i was signi®cantly elevated above that

evoked by carbachol alone (Fig. 6b). Therefore the d-opioid

receptor does not exhibit cross-talk with the sphingosine

kinase-coupled LPA receptor in SH-SY5Y cells.

Potassium channel activation

Opioid receptors activate several different types of potas-

sium conductance (Wimpey and Chavkin 1991; North 1993;

Vaughan et al. 1997). Although most of the elevation of

Fig. 5 Effect of UTP and DPDPE on [Ca21]i and IP1 production in

CHO-d cells. (a) The trace represents a continuous record of [Ca21]i

in a monolayer of CHO-d cells. DPDPE alone did not elevate [Ca21]i

whereas UTP produced a biphasic elevation of [Ca21]i. In the

presence of UTP, DPDPE now also elevated [Ca21]i. Drugs were

applied for the periods indicated by the bars. (b) Concentration±

response relationships for UTP-evoked elevation of [Ca21]i (open

squares) and accumulation of IP1 (closed squares) in CHO-d cells.

The data represent the mean ^SEM from 4 to 6 experiments. (c)

Time course of IP1 accumulation in CHO-d cells under the following

conditions: (i) basal (A), (ii) in the presence of DPDPE (1 mM) (B),

(iii) in the presence of UTP (10 mM) (K), and (iv) in the presence of

UTP (10 mM) plus DPDPE (1 mM) (O). Each point represents the

mean ^SEM of 3±7 experiments; * indicates a signi®cant difference

(p , 0.05) over UTP alone.

Co-incident signalling between d-opioid and m3 muscarinic receptors 1695

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1688±1700

[Ca21]i evoked by d-opioid receptor activation resulted from

Ca21 release from intracellular stores, part of the elevation

of [Ca21]i could be secondary to increased potassium con-

ductance and membrane hyperpolarization that would

increase the driving force for Ca21 entry through capacitative

Ca21 entry channels opened on store depletion during Gq-

coupled muscarinic receptor activation (Grudt et al. 1996).

In whole cell patch clamp recordings from single SH-SY5Y

cells application of DPDPE (1 mm) did not activate an

inwardly rectifying potassium current nor did it modify the

voltage-activated potassium current (Fig. 7). In SH-SY5Y

cells there is little evidence of an inwardly rectifying mem-

brane conductance. Since the inwardly rectifying potassium

conductance would be most readily observed as inward

current we stepped cells to potentials negative to EK.

The current at 2120 mV/was8.0 ^ 5.1 pA in control and

215.7 ^ 6.4 pA in the presence of DPDPE (1 mm,

p . 0.05, n � 4). Therefore membrane hyperpolarization

as a result of an increase in potassium current was not

involved in the d-opioid receptor-induced elevation of

[Ca21]i.

Elevation of [Ca21]i by DPDPE in cell suspensions

In stirred suspensions of SH-SY5Y cells the 340/380 nm

ratio increased throughout the period of recording (Fig. 8).

This is in contrast to the stable 340/380 nm ratio observed

with monolayers of cells (see Fig. 1). When added to stirred

suspensions of SH-SY5Y cells, DPDPE (1 mm) alone caused

an elevation of [Ca21]i (Fig. 7). Pretreatment of suspensions

of SH-SY5Y cells with apyrase (3 U/mL for 45 min), to

degrade extracellular ATP, ADP, UTP and UDP, reduced

the response to DPDPE (1 mm) by 62 ^ 13% (p , 0.05,

n � 5). It is unlikely that apyrase is degrading DPDPE

because in monolayer experiments addition of apyrase did

not affect the DPDPE response (data not shown). These

results indicate that in cell suspensions there may be con-

siderable release into the extracellular medium of endogen-

ous agonists at Gq-coupled receptors, presumably as a result

of cell damage, and that it was the Gq-coupled receptor

activation which revealed the Gi/Go-coupled receptor-

mediated response.

Fig. 6 Lack of cross-talk between d-opioid receptors and LPA

receptors in SH-SY5Y cells. (a) The trace represents a continuous

record of [Ca21]i in a monolayer of SH-SY5Y cells. DPDPE alone

did not elevate [Ca21]i (data not shown) whereas LPA produced a

transient elevation of [Ca21]i that declined back to basal levels even

in the continuous presence of LPA. In the presence of LPA, DPDPE

did not elevated [Ca21]i. Drugs were applied for the periods indi-

cated by the bars. (b) Co-application of LPA (1 mM) plus DPDPE

(1 mM) for 1 min did not elevate [Ca21]i above that observed with

LPA alone. In contrast co-application of carbachol (1 mM) plus

DPDPE (1 mM) for 1 min did elevate [Ca21]i above that seen with

carbachol alone. In each experiment the amplitude of the responses

has been normalized to that evoked by an initial application of carba-

chol (1 mM) alone. Each column represents the mean and SEM of 5

experiments; *indicates a signi®cant difference (p , 0.05) over the

appropriate control.

Fig. 7 d-Opioid receptors did not activate an inwardly rectifying

potassium conductance in SH-SY5Y cells. Current-voltage relation-

ships in the absence (O) and presence of DPDPE (1 mM) (K).

Currents were elicited by 40 ms pulses from a holding potential of

260 mV. Each point represents the mean ^SEM of 5 experiments.

1696 A. Yeo et al.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1688±1700

Discussion

In SH-SY5Y neuroblastoma cells, as in other neuronal and

non-neuronal cell types, activation of various Gi/Go-coupled

receptors can elevate [Ca21]i but only if there is concomi-

tant activation of a Gq-coupled receptor (Gerwins and

Fredholm 1992; Okajima and Kondo 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; Selbie and Hill 1998; Toms and Roberts

1999; Cho et al. 2000). We have chosen in this study to

examine the interaction between the Gi/Go-coupled d-opioid

receptor and the m3 muscarinic receptor because in SH-

SY5Y cells, co-application of agonists at each of these

receptors evoked a robust, coincident elevation of [Ca21]i.

The coincident signalling required concomitant activation

of both receptor types because it was abolished when a

muscarinic receptor antagonist was applied simultaneously

with the d-opioid receptor agonist (Connor and Henderson

1996). The d-opioid receptor-mediated component was

abolished by pretreating the cells with pertussis toxin demon-

strating that activation of Gi/Go proteins was involved.

Furthermore, the coincidence was speci®c between Gi/Go-

and Gq-coupled receptors in that activation of the LPA

receptor did not reveal the d-opioid receptor-mediated

response. In SH-SY5Y cells LPA did not stimulate inositol

phosphate turnover but elevated [Ca21]i by stimulating a G

protein-coupled receptor that, by an as yet unknown mech-

anism, activated sphingosine kinase and thus elevated [Ca21]i

(Young et al. 1999). We can also rule out coupling of the Gi/

Go-coupled d-opioid receptor to sphingosine kinase to elevate

[Ca21]i in SH-SY5Y cells because the d-opioid receptor-

mediated response was not inhibited by a sphingosine kinase

inhibitor, indeed it was potentiated which may indicate that

part of the d-opioid receptor-elevation of [Ca21]i pathway is

under the negative control of sphingosine kinase.

bg dimers released from Gi/Go proteins on d-opioid

receptor activation play a key role in the elevation of [Ca21]i

because the elevation was abolished when free bg dimers

were sequestered by overexpression of the bg binding

domain of GRK2 (Koch et al. 1993). On the other hand the

m3 muscarinic, Gq-mediated elevation of [Ca21]i was

unaffected. Free bg dimers have been shown to directly

activate PLCb isozymes (Park et al. 1993; Smrcka and

Sternweiss 1993; Rhee and Bae 1997) but the isozymes

are not equally sensitive to stimulation by bg dimers. The

order of sensitivity of PLC isozymes to bg stimulation is

PLCb3 . PLCb2 . PLCb1. Free bg dimers from Gi/Go

were not suf®cient to stimulate PLC in SH-SY5Y cells

because activation of d-opioid receptors alone did not either

enhance inositol phosphate turnover or elevate [Ca21]i. This

may indicate that in SH-SY5Y cells PLCb1 is the pre-

dominant form of the enzyme expressed. We have detected

PLCb1, PLCb2 and PLCb3 in lysates of SH-SY5Y cells

using the Western blotting technique (Yeo and Henderson,

unpublished observations) but have been unable to quantify

their respective levels of expression. Elevation of [Ca21]i

was only observed with concomitant Gi/Go-and Gq-coupled

receptor activation. Therefore bg dimers from Gi/Go were

crucial to this coincident signalling but were not on their

own suf®cient to elevate [Ca21]i.

To give rise to coincident signalling bg dimers released

on Gi/Go-coupled receptor activation and aq dimers acti-

vated by Gq-coupled receptor activation may both bind to

PLC and thus produce a synergistic activation (Chan et al.

2000; Zhu and Birnbaumer 1996; Selbie et al. 1997; Tomura

et al. 1997; Selbie and Hill 1998). Furthermore, bg dimers

have been shown to decrease phosphorylation of PLCb by

PKC (Litosch 1997) which could delay the onset of negative

feedback by PKC and prolong PLCb activation. Given that

such synergy at the level of PLC should result in enhanced

inositol phosphate production as well as enhanced elevation

Fig. 8 Apyrase reduced the elevation of [Ca21]i by DPDPE alone in

stirred suspensions of SH-SY5Y or CHO-d cells. (a) The traces

represent continuous records of [Ca21]i in stirred suspensions of

SH-SY5Y cells. Addition of DPDPE (1 mM) evoked an elevation of

[Ca21]i. The response evoked by DPDPE was reduced when the cell

suspension had been treated with apyrase (3 U/mL). The traces are

representative of data obtained in 5 separate experiments. (b) The

traces represent continuous records of [Ca21]i in stirred suspensions

of CHO-d cells. Addition of DPDPE (1 mM) evoked an elevation of

[Ca21]i. The response evoked by DPDPE was reduced when the cell

suspension had been treated with apyrase (3 U/mL). The traces are

representative of data obtained in 3 separate experiments.

Co-incident signalling between d-opioid and m3 muscarinic receptors 1697

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1688±1700

of [Ca21]i this mechanism cannot explain our ®ndings in

SH-SY5Y cells where the elevation of [Ca21]i occurred in

the absence of enhanced inositol phosphate production. It

would, however, explain our results from CHO-d cells where

enhanced inositol phosphate production was observed.

Could we, for technical reasons, have missed d-opioid

receptor-mediated enhancement of inositol phosphate pro-

duction in SH-SY5Y cells? Dickenson and Hill (1998)

demonstrated for the Gi/Go-coupled 5-HT1B receptor in

CHO cells that elevation of [Ca21]i, but not inositol

phosphate production, could be observed at low levels of

receptor expression but that at higher levels of receptor

expression both an elevation of [Ca21]i and an increase in

inositol phosphate production could be observed. We feel,

however, that it was unlikely that we were missing an

enhancement of inositol phosphate production in SH-SY5Y

cells for the following reasons. First, we used concentrations

of DPDPE and oxo-M which produced similar peak

elevations of [Ca21]i and could measure enhanced inositol

phosphate production only in response to oxo-M but not

DPDPE. Second, the proportion of cells responding and the

amplitude of the elevations of [Ca21]i by DPDPE in SH-

SY5Y and CHO-d cells was similar but we could only

measure enhanced inositol phosphate production in CHO-d

cells not in SH-SY5Y cells. Given that bg and aq dimers

exert an additive stimulatory effect on PLCb3 but not

PLCb1 (Smrcka and Sternweiss 1993), an interaction at the

level of PLC to enhance inositol phosphate production

would not be observed in SH-SY5Y cells if the predominant

isozyme of PLC expressed in these cells was PLCb1.

Neuronal cells express higher amounts of Gi and Go than

Gq (Hepler and Gilman 1992) and excess bg dimers from

activated Gi/Go proteins could associate with free aq dimers

in their GDP bound state to replenish the supply of Gq. In

the continued presence of ongoing Gq-coupled receptor

activation, replenishment of the available Gq would result in

more rapid production of active aq dimers in their GTP

bound state and thus enhanced PLC activation (Quitterer and

Lohse 1999). However, this mechanism for Gi/Go-and Gq-

coupled receptor cross-talk should also be revealed as an

enhancement of inositol phosphate production and therefore

is unlikely to apply in SH-SY5Y cells.

The m3 muscarinic receptor desensitized on continuous

agonist exposure (Szekeres et al. 1998). We can discount the

possibility that Gi/Go-coupled receptor activation in some

way reversed the desensitization of the Gq-coupled m3

muscarinic receptor for two reasons. First, enhancement of

the muscarinic response through reduced desensitization

would again result in enhancement of inositol phosphate

production which we did not observe. Second, the amplitude

of the d-opioid receptor-mediated elevation in [Ca21]i was

equal in amplitude at various time points during the

muscarinic receptor-mediated response whereas desensitiza-

tion would have been expected to increase with time.

It has been suggested that activated G proteins may in

some way sensitize the IP3 receptor to IP3 (Xu et al. 1996;

Neylon et al. 1998) thus enhancing Ca21 release from

intracellular stores and that this effect was mediated by free

bg dimers (Zeng et al. 1996). With this mechanism the

cross-talk between Gi/Go-and Gq-coupled receptors would

occur at the level of the IP3 receptor, not at the level of PLC,

and would give rise to an elevation of [Ca21]i without an

enhancement of inositol phosphate production. The IP3

receptor is a tetrameric ligand-gated ion channel with four IP3

binding sites (Taylor et al. 1999). An interaction between bg

dimers and IP3 receptors could explain why the concentra-

tion±response curve for carbachol's permissive action to

reveal the d-opioid receptor response is shifted to the left.

There is some controversy surrounding the d-opioid

receptor-mediated elevation of [Ca21]i in the NG108-15

neuroblastoma � glioma hybrid cell line. We and colleagues

have demonstrated that, in that cell line, as in SH-SY5Y cells,

d-opioid receptor-mediated elevation of [Ca21]i required

concomitant Gq-coupled receptor activation (Okajima and

Kondo 1992; Okajima et al. 1993; Connor et al. 1994).

However, Thayer and colleagues have reported that in

NG108-15 cells d-opioid receptor activation alone was

suf®cient to increase [Ca21]i (Jin et al. 1994; Strassheim

et al. 1998; Yoon et al. 1999). It is possible that under

different culture conditions in different laboratories NG108-

15 cells may express different PLCb isoforms. If under their

culture conditions NG108-15 cells expressed PLCb3 then

on d-opioid receptor activation free bg dimers could directly

activate PLCb3, generate IP3 and release Ca21 from intra-

cellular stores.

In our single cell imaging experiments we observed a

rise in [Ca21]i in response to d-opioid receptor activation

in only 5% of cells, whereas 60% of cells responded to d-

opioid receptor activation during concomitant m3 muscarinic

receptor activation. Most neuronal cell lines are not entirely

homogeneous. We have evidence that SH-SY5Y cells are

somewhat heterogeneous with respect to their expression of

d-and m-opioid receptors. In single cell imaging experi-

ments, in the presence of concomitant m3 muscarinic

receptor activation, there were cells which responded to

d-opioid receptor activation, some to m-opioid receptor

activation but the majority responded to both (Yeo and

Henderson, unpublished observations). If a small population

of SH-SY5Y cells expressed predominantly PLCb3 then

these cells might exhibit a rise in [Ca21]i in response to

d-opioid receptor activation alone. In addition, we cannot

exclude the possibility that those cells responding to

d-opioid receptor activation alone were not being concomi-

tantly activated by endogenous Gq receptor agonists

released into the bathing medium from the cells themselves

or from neighbouring cells. Even though we used a fast

superfusion rate (4 mL/min) we cannot be sure that we had

rapid ¯uid exchange in the spaces between adjacent cells or

1698 A. Yeo et al.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1688±1700

in the space between the cells and the coverslip on which

they were grown. Our observations on stirred cell suspen-

sions, where concomitant m3 muscarinic receptor activation

was not required to see a d-opioid receptor elevation of

[Ca21]i and apyrase markedly depressed the response to

d-opioid receptor activation alone, indicate that with this

method there was considerable release into the extracellular

medium of endogenous agonists at Gq-coupled receptors

presumably as a result of cell damage and that it was the

Gq-coupled receptor activation which revealed the Gi/Go-

coupled receptor-mediated response.

In neuronal and non-neuronal cells activation of Gi/Go-

coupled receptors has been reported to elevate [Ca21]i by

enhancing Ca21 entry into the cell through dihydropyridine-

sensitive, presumably l-type voltage-operated, Ca21 channels

(Jin et al. 1992; Eriksson et al. 1993; Tang et al. 1994; Smart

et al. 1995; Wandless et al. 1996). We have no experimental

evidence for any L channel component to the d-opioid

receptor-mediated elevation of [Ca21]i in SH-SY5Y cells. In

our hands the rise in [Ca21]i persisted in the absence of

extracellular Ca21 and was not reduced by nimodipine or

La31, agents which block l-type Ca21 channels (Connor

and Henderson 1996). Furthermore, m3 muscarinic receptor

activation and d-opioid receptor activation released Ca21

from the same thapsigargin-sensitive intracellular Ca21

store (Connor and Henderson 1996; this paper). Under our

culture conditions SH-SY5Y cells expressed few l-type

channels although, on prolonged depolarization, a rise in

[Ca21]i due to Ca21 entry through l-type channels could be

observed (Seward and Henderson 1990; Morton et al. 1992).

Another Ca21 entry pathway in SH-SY5Y cells is through

capacitative Ca21 entry pathways (Grudt et al. 1996).

d-opioid receptor activation did not however, modify this

Ca21 entry process (Connor et al. 1995).

In conclusion, the coincident signalling between d-opioid

and m3 muscarinic receptors in SH-SY5Y cells involves bg

subunits released from Gi/Go on d-opioid receptor activa-

tion. The site of interaction does not appear to be PLC but at

a step after PLC activation.

Acknowledgements

Single cell imaging experiments were carried out in the MRC

Cell Imaging Facility at the University of Bristol. We thank Dr

R. Lefkowitz for the gift of the plasmid containing the C

terminal region (amino acids 495±689) of GRK2, Dr L. Devi

for the gift of CHO-d cells, Prof. J. Tavare for advice and

facilities for plasmid preparation and Dr M. Connor for helpful

discussions.

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