RGS9 proteins facilitate acute tolerance to mu-opioideffects

Javier GarzoÂn, Marta RodrõÂguez-DõÂaz, Almudena LoÂpez-Fando and Pilar SaÂnchez-BlaÂzquezNeurofarmacologõÂa, Instituto de NeurobiologõÂa Santiago RamoÂn y Cajal, Consejo Superior de Investigaciones Cientõ®cas,

Avenida Doctor Arce 37, E-28002 Madrid, Spain

Keywords: 125I-Tyr27-human b-endorphin, analgesia, antinociception, antisense technology, G-proteins, mouse, receptordesensitization, regulator of G-protein signalling

Abstract

This paper reports that regulators of G-protein signalling (RGS) proteins modulate the timing and amplitude of opioid signals by a

push±pull mechanism. This is achieved without noticeable changes in the binding properties of opioids, e.g. b-endorphin to mu-

opioid receptors. The expression of RGS proteins was reduced by blocking their mRNA with antisense oligodeoxynucleotides(ODN). Knock down of RGS2 or RGS3 diminished morphine and b-endorphin analgesia, whereas that of RGS9 or RGS12

enhanced this activity. In mice with impaired RGS9, but not impaired RGS2, the potency and, in particular, the duration of opioid

antinociception increased. Further, the animals did not exhibit acute tolerance generated by a single and ef®cacious dose of

morphine, nor did they develop tolerance after a daily i.c.v. injection of the opioid for 4 days. In a model of sustained morphinetreatment, the impairment of RGS9 proteins facilitated increases in the response to the delivered opioid. This was only effective

for 2±3 h after the subcutaneous implantation of an oily morphine pellet; later, tolerance developed. To reduce the impact of the

chronic morphine acting on opioid receptors, other RGS proteins presumably substitute the GTPase-activating function of RGS9on morphine-activated Ga-GTP subunits. The desensitization of mu-opioid receptors appears to be a cell membrane-limited

process facilitated by RGS9¢s sequestering of agonist-segregated Ga subunits.

Introduction

Normally, repeated administration of opioids bring about a progres-

sive decrease in their potency. This is observed after even single

doses of agonists (acute tolerance or tachyphylasis), and after long-

term treatments that lead to development of tolerance and physical

dependence on these substances. Signi®cant knowledge has been

accumulated on the adaptive alterations triggered by the chronic use

of opioids, including the gating of ion channels, the expression of

particular genes, changes in the number and function of guanine

nucleotide-binding regulatory protein (G-proteins) (Strassheim &

Malbon, 1994), marked up-regulation of the adenylyl cyclase/cAMP

system, enhancement of (cAMP)-dependent protein kinase (PKA)

(Nestler et al., 1993) and PKC activities (Narita et al., 1994), as well

as modi®cations in cell morphology (see, e.g. Nestler & Aghajanian,

1997). However, there have been fewer studies on tolerance induced

by an adequate single dose of opioids. The threshold dose necessary

to produce acute tolerance is about three to four times greater than

that required for producing analgesia (Huidobro et al., 1976). This

interesting phenomenon of acute tolerance appears within hours of

agonist administration and lasts for at least 2±3 days (Huidobro-Toro

& Way, 1978). It has been proposed that the mechanisms responsible

for acute tolerance parallel those underlying chronic morphine

tolerance, as both situations can be modulated by identical agents

(Fairbanks & Wilcox, 1997). In fact, tolerance to single doses of

opioids is accompanied by some degree of physical dependence that

is milder than that observed in the chronic morphine-dependent state

(Huidobro-Toro & Way, 1978; Bilsky et al., 1996). Pharmacological

studies have shown the possibility of regulating opioid-induced acute

tolerance (see, e.g., Fairbanks & Wilcox, 1997). However, precise

information is lacking on the role of opioid-acted transduction on the

genesis and timing of the changes responsible for acute tolerance or

tachyphylaxis.

Our knowledge of the particular elements involved in the

internalization and processing of the extracellular signals has greatly

increased in recent years. Besides receptors, G-proteins and effectors,

regulator of G-protein signalling (RGS) proteins have been described

as cooperating in the internalization of agonist-initiated messages. To

date, > 20 proteins are reported to contain RGS domains. The

majority are expressed in brain and differ in size, amino-acid

composition and distribution (Gold et al., 1997; Hepler, 1999). RGS

proteins serve as GTPase-activating proteins (GAPs) for a subunits of

heterotrimeric Gi, Go, Gz, Gt and Gq proteins (Berman & Gilman,

1998). By activating the metabolism of GTP to GDP, RGS proteins

promote the Ga-GDP state capable of binding to Gbg dimers

(Berman & Gilman, 1998; Hepler, 1999). However, during the period

which RGS proteins remain bound to Ga subunits, Ga(GDP)-Gbg re-

association is delayed. Thus, RGS proteins emerge as potential

negative regulators of G-protein signalling. The present study

addresses the role of RGS proteins in the potency of opioids, the

development of tachyphylasis to their effects, and long-term toler-

ance. With this purpose, oligodeoxynucleotides (ODNs) to mRNAs

coding for the described murine RGS proteins were given to mice,

and antinociception was taken to represent a paradigmatic opioid

Correspondence: Dr Javier GarzoÂn, as above.E-mail: jgarzon@cajal.csic.es

Received 31 July 2000, revised 27 October 2000, accepted 11 December 2000

European Journal of Neuroscience, Vol. 13, pp. 801±811, 2001 ã Federation of European Neuroscience Societies

effect. The results show an important role for RGS9 proteins in the

development of acute tolerance to opioids at mu-receptors.

Materials and methods

Reduction of RGS function

Synthetic end-capped phosphorothioate ODNs were prepared by solid

phase phosphoramidite chemistry using a CODER 300 DNA

synthesizer (DUPONT; Wilmington, DE, USA) at the 1-mmol

scale. The introduction of phosphorothioate linkages was achieved

by tetraethylthiuram disulphide sulphurization. Crude ODNs were

puri®ed by conventional reverse-phase chromatography through a 5-

mm/C18 column (Spherisorb ODS-2, 150 3 4.6 mm, Symta, Madrid,

Spain) using 0.1 M triethylammonium acetate (pH 7.0) and aceto-

nitrile as the mobile phase. To reduce the synthesis of RGS proteins,

the following ODN sequences were used (PrimerSelect, DNASTAR

Inc, Madison, WI, USA):

FIG. 1. Knock down of RGS proteins andmorphine antinociception. Animals receivedeither saline, the ODN-RD or the antisenseODN into the right lateral ventricle over a 5-day schedule. On day 6, 3 nmol morphine wasi.c.v.-injected and antinociceptive activityevaluated 30 and 90 min later. The opioidagonist produced similar effects in mice thati.c.v.-received the vehicle or the random oligo,and in noninjected (naive) animals. Thus, theresponsiveness of the mice was not altered bythe experimental procedure alone. Values arethe mean 6 SEM from groups of 10 mice.*P < 0.05 vs. the control group, ANOVA,Student±Newman±Keuls test. (Lower panels)Detection of RGS9-2 and RGS2 in mousebrain. The ef®cacy and selectivity of the ODNtreatments was assessed with antibodiesdirected to RGS2 proteins, RGS9 proteins,Gb1/2 subunits, Gi2a subunits and mu-opioidreceptors. SDS-solubilized cerebral cortex,striatum and PAG were resolved by SDS±PAGE (10±20% acrylamide/2,6%bisacrylamide) and Western blotted.Immunodetection was carried out as describedin Materials and Methods. Detection of RGS9-2 mRNA using RT-PCR. Lanes contain 0.3 mgof total RNA from the mouse brain structures.

802 J. GarzoÂn et al.

ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 13, 801±811

the 16-base ODN 5¢-C*T*CTATGAAGTCGG*T*A-3¢ to nucleotides

428±443 of the RGS2 gene (ODN1),

the 16-base ODN 5¢-G*C*TGAATTCAAGGT*C*A-3¢ to nucleotides

743±758 of the RGS2 gene (ODN2),

the 16-base ODN 5¢-C*A*CAGCATTCGGTC*C*A-3¢ to nucleotides

1211±1227 of the RGS2 gene (ODN3) (U67187),

the 16-base ODN 5¢-C*T*TCATGTCCTTGG*C*T-3¢ to nucleotides

105±120 of the RGS3 gene (AA880930),

the 17-base ODN 5¢-G*G*ACTTAGTTTAGAA*G*G-3¢ to nucleo-

tides 310±326 of the RGS4 gene (Ab004315),

the 16-base ODN 5¢-C*T*GTCTGGATGAAT*T*C-3¢ to nucleotides

389±404 of the RGS5 gene (U67188),

the 17-base ODN 5¢-G*G*TCTTGTCATAACT*C*T-3¢ to nucleo-

tides 1381±1397 of the RGS7 gene (Af011360),

the 16-base ODN 5-T*G*GATCAACATAGA*C*G-3¢ (ODN1) to

nucleotides 532±547 of the RGS9 gene,

the 16-base ODN 5¢-C*T*CGAATCAGTTCG*C*T-3¢ to nucleotides

973±988 of the RGS9 gene (ODN2),

the 16-base ODN 5¢-C*G*CAGACCCACATT*T*A-3¢ to nucleotides

1234±1249 of the RGS9 gene (ODN3) (Af011358),

the 16-base ODN 5¢-C*T*ACAGGCCTCTGA*C*A-3¢ to nucleotides

165±180 of the RGS12 gene (AA855540),

the 16-base ODN 5¢-G*T*AGTGGCAGCTTG*T*A-3¢ to nucleotides

431±446 of the RGS14 gene (U70427) and

an 18-base ODN 5¢-A*T*GTCCGTCTGTCCTT*C*C-3¢ to nucleo-

tides 832±850 of the RGS16 gene (U67189).

These sequences displayed no homology to other relevant cloned

proteins (GeneBank database). Antisense ODN controls consisted of

mismatched sequences in which some bases were switched (in bold)

without altering the remaining sequence:

RGS2M1, 5¢-C*A*CTAAGATGTCCGAA-3¢;RGS2M2, 5¢-G*C*AGAATACATGGA*C*A-3¢;RGS2M3, 5¢-CACTCCATACGCTGCA-3¢; and

RGS9M1, 5¢-T*C*GATGAACTAAGT*G*C-3¢;RGS9M2, 5¢-C*T*GCAATGAGTTGC*T*C-3¢;RGS9M3, 5¢-C*G*TACACGCACTTA*T*A-3¢;

and also of a random ODN (ODN-RD) (SaÂnchez-BlaÂzquez et al.,

1995).

ODN solutions were made up in saline immediately prior to use.

Different control groups of animals were used to ascertain the

speci®city of ODN treatments. These controls include noninjected

mice (naive), those that received the vehicle (saline), and animals

injected with a random sequence (ODN-RD) or mismatched antisense

sequence (ODN-RGS#M). As no signi®cant differences were

observed in the response to the analgesic substances of naive,

vehicle-, ODN-RD- or ODN-RGS#M-injected mice (see e.g. Figs 1

and 2), only the data from the control ODNs (ODN-RD or ODN-

RGS#M) are presented in this paper.

Animals were lightly anaesthetized with ether and injections made

into the lateral ventricle with a 10-mL Hamilton syringe to a depth of

3 mm, 2 mm lateral and 2 mm caudal to the bregma. The 4-mL

content was infused at a rate of 1 mL every 5 s. The needle was

maintained for an additional 10 s. To minimize the chance of

neurotoxicity caused by repeated i.c.v. injections, an interval of 24 h

was allotted between successive administrations of the ODNs

(Chiasson et al., 1994). Each ODN treatment was performed on a

distinct group of mice according to the following 5-day schedule: on

days 1 and 2 with 1 nmol, on days 3 and 4 with 2 nmol, on day 5 with

3 nmol (SaÂnchez-BlaÂzquez et al., 1995; GarzoÂn et al. 2000).

Functional studies usually started on day 6. The possible alterations

promoted by ODN treatments on animal activity were recorded with a

Digiscan animal activity monitor system (activity cage) (Omnitech

electronics, Columbus, OH, USA). Groups of four mice that had

received an ODN following the above-described schedule were

monitored for horizontal and vertical activity for periods of 10 min.

Only ODNs that produced no signi®cative changes in these

behavioural parameters were selected for this study (ANOVA,

Student±Newman±Keuls test, P < 0.05).

To monitor any possible injury to tissue structure caused by i.c.v.

delivery of the ODNs, Nissl staining was routinely performed on

brain coronal slices from mice receiving each ODN treatment. These

slices included some of the periventricular regions responsible for the

antinociceptive effects of opioids injected via the i.c.v. route (Yaksh

et al., 1976; Rossi et al., 1994). The histological procedure is

described elsewhere (GarzoÂn et al. 2000). The ODNs produced no

alterations to the normal structure of periaqueductal grey matter

(PAG) or any other periventricular region (not shown).

Detection of RGS9 and RGS2 in mouse brain: electrophoresisand immunoblotting

At the end of ODN treatment, groups of mice were killed by

decapitation. The animals' cerebral cortices, striata and PAG were

then collected. For each determination, the structures from four mice

were pooled and SDS-solubilized P2 membranes were subsequently

resolved by SDS/polyacrylamide gel electrophoresis (PAGE) in

8 3 11 3 1.5 cm gel slabs (10±20% total acrylamide concentration/

2.6% bisacrylamide cross-linker concentration). For RGS9 immuno-

detection, 45, 60 and 80 mg protein/lane were used for striatum, PAG

and cerebral cortex determination, respectively. For RGS2 immuno-

detection, 60 mg protein/lane were used for each neural structure.

Afterwards, the separated proteins were transferred to 0.2-mm

polyvinylidene di¯uoride membranes (Bio-Rad). Polyclonal anti

RGS2 and anti RGS9 1 : 500 [Santa Cruz Biotechnology, Inc.,

Santa Cruz, CA, USA; SC9103 (rabbit) and SC8143 (goat),

respectively,], anti Gi2a subunits (SaÂnchez-BlaÂzquez et al., 1995),

anti Gb1/2 subunits (Lang & Costa, 1987) and anti mu-opioid

receptor (SaÂnchez-BlaÂzquez et al., 1995, 1997) 1 : 1000 were diluted

in TBS-0.05% Tween 20 (TTBS) and incubated with the transfer

membranes at 6 °C for 24 h. The SC8143 was detected with donkey

antigoat IgG horseradish peroxidase conjugate antiserum (Santa Cruz

Biotechnology, SC2020). The other polyclonal antisera were detected

with a goat antirabbit IgG (H+L) horseradish peroxidase conjugate

antiserum (BioRad; 170±6515, Madrid, Spain). Secondary antisera

were diluted 1 : 2000 in TTBS, incubated for 3 h and visualized with

the DAB substrate solution. Immunoblots were analysed by

densitometry using an instrument with re¯ectance capabilities (GS-

700 Imaging Densitometer, BioRad) and Molecular Analyst Software

(v 1.5; BioRad).

RT-PCR

Total RNA was harvested from mouse brain structures using a single-

step procedure (Ultraspec RNA isolation system, Biotecx Labs,

Houston, TX, USA), based on the formation of RNA complexes with

guanidinium molecules, followed by isopropanol precipitation. The

pellet was washed in 75% ethanol, dried, resuspended in 40 mL RNA

storage solution (Ambion, Austin, TX, USA) and stored at ±80 °C

until analysis. The yield of RNA was determined using UV

spectrometry (260 nm).

Two micrograms of total striatum, PAG and cerebral cortex RNA

was reverse transcribed using the RT-PCR First Strand Synthesis Kit

(RETROscript, Ambion) with oligo (deoxythymidine) priming.

cDNA synthesis was carried out at 42 °C for 60min. PCR reactions

were performed on 3, 5 and 7 mL of RT product (equivalent to 0.3,

0.5 and 0.7 mg total RNA, respectively) in a ®nal volume of 50 mL of

RGS9 facilitates opioid tachyphylaxis 803

ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 13, 801±811

the buffer solution containing 10 mM Tris HCl (pH 8.3), 50 mM KCl,

1.5 mM MgCl2, 125 mM of each dNTP, 0.5 mM of each primer and

1 unit of SuperTaq Thermostable DNA polymerase (Ambion). cDNA

primers (AmershamPharmacia Biotech, Barcelona, Spain) directed

towards the murine RGS9 gene (AF011358) were adapted from those

described by Granneman et al. (1998) directed towards rat and human

counterparts. The sequences were: forward 5¢-GAAGCCTGTGAGG-

ACCTGAAGTACG-3¢ (sense) corresponding to base pairs 1060±

1084, and reverse 5¢-AGGAGGCAGCTCCTTTTTGAGTTG-3¢(antisense) corresponding to base pairs 1498±1521 (in order to

yield an expected band size of 462 base pairs). The RT-PCR products

were initially denatured at 95 °C for 1 min and then subjected to

cycles of denaturation (95 °C, 20 s) and annealing (65 °C, 30 s).

Ampli®cation cycles were 35 in total. After the last, extension was

performed for 7 min at 65 °C. A DNA Thermal Cycler 480 (PE

Biosystems, Foster City, CA, USA) and thin-walled 0.5 mL PCR

tubes (Ambion) were used. PCR products were electrophoresed

(34 V, 2.5 h) in a 1.5% agarose gel with PCR markers 50±2000

(Novagen, Madison, WI, USA), and then incubated in SYBR gold

solution (Molecular Probes, Poortgebouw, Netherlands) for 20 min.

FIG. 2. The in¯uence of RGS9 proteins on the time course of morphine-evoked analgesia. Animals with reduced levels of RGS9 proteins throughODN treatment were i.c.v.-injected with various doses of morphine andanalgesia was determined at various intervals by the warm water 52 °C tail-¯ick test. The effects of the opioid at 30, 60 and 90 min post injection arealso presented in dose-effect curves. Values are mean 6 SEM from groupsof 10±20 mice. *P < 0.05 vs. the group that had received the mismatchedcontrol ODN-RGS9M or the vehicle (saline).

FIG. 3. Opposite effects of RGS9 and RGS2 proteins on the time course ofb-endorphin-evoked analgesia. *,+P < 0.05 vs. the group that had receivedthe mismatched control ODN-RGS9M or ODN-RGS2M, respectively.Details as in Fig. 2.

804 J. GarzoÂn et al.

ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 13, 801±811

DNA products were visualized with UV light (Gelprinter, T.D.I.,

Madrid, Spain).

Preparation of membranes from mouse brain

Male albino CD/1 mice (Charles River, Barcelona, Spain) weighing

22±27 g were used to provide experimental tissue. Mice that had

received i.c.v. injections of the ODNs were killed by cervical

dislocation. Brains were quickly removed and washed in ice-cold

50 mM Tris HCl, 0.32 M sucrose, pH 7.5, at 4 °C. PAGs (up to 1 mm

around the aqueduct) were collected and homogenized using a

Polytron (model PT 10±35, Kinematica, Kriens-Luzern, Switzerland)

at setting of 3 for 15 s. The homogenate was then centrifuged at

1000 g at 4 °C for 10 min (Sorvall RC5C, rotor SS-34 Newton, CT,

USA). After the pellet was discarded, the supernatant was centrifuged

at 20 000 g for 20 min. The pellet was resuspended in buffer and

centrifuged at 20 000 g for an additional 20 min. The ®nal pellet (P2)

was diluted in Tris buffer supplemented with a mixture of protease

inhibitors (0.2 mM phenylmethylsulphonyl ¯uoride, 2 mg/mL leu-

peptin and 0.5 mg/mL aprotinin) to a ®nal protein concentration of

» 2 mg/mL.

Binding studies125I-Tyr27 human b-endorphin, obtained lyophilized, was reconsti-

tuted with 100 mL of distilled water to give 0.25% BSA, 5%

lactose, 0.2% L-cysteine hydrochloride, 10 mM citric acid, and

800 KIU/mL aprotinin. Four-millitre siliconized borosilicate glass

tubes (Sigmacote from Sigma, St Louis, MO, USA) were used

(adsorption of the iodinated peptide was reduced to < 5%). To

restrict the speci®c binding of iodinated b-endorphin to only mu-

opioid receptors, saturation assays of 125I-Tyr27 human b-

endorphin, used at concentrations ranging from 0.3 pM to 3 nM,

were conducted in the presence of 300 nM N,N-diallyl-Tyr-(a-

aminoisobutyric acid)2-Phe-Leu-OH (ICI-174,864; GarzoÂn et al.,

1998). The membrane suspension reached a ®nal concentration of

» 0.2 mg/mL, 0.2% BSA, 0.01% bacitracin, incubation volume

was made up to 2 mL with 50 mM (®nal concentration) Tris HCl

buffer, pH 7.5. Non-speci®c binding was assessed in the presence

of 1 mM unlabelled b-endorphin. Samples were incubated in

triplicate at 25 °C for 90 min in a shaking incubator and ®ltered

under vacuum (Harvester M-12R, Brandel Gaitherburg, Maryland,

USA) through glass ®bre disks (Whatman GF/B, Maidstone,

England) previously immersed for 3 h in 5 mM Tris HCl/0.3%

polyethylenimine (Sigma), to minimize binding to the ®lters. The

®lters were then washed three times with 4 mL of ice-cold 5 mM

Tris HCl buffer, pH 7.5, placed in polyethylene counting vials

and counted using a LKB Compugamma CS counter (Bromma,

Sweden).

Animals and evaluation of antinociception

Male albino mice CD-1 weighing 22±25 g were housed and used

strictly in accordance with the guidelines of the European Community

for the Care and Use of Laboratory Animals (Council Directive 86/

FIG. 4. Role of RGS9 on acute tolerance to a single dose of morphine.Saline or a priming dose of 10 nmol morphine was i.c.v.-injected to themice that had received the ODN2 to RGS9, the mismatched ODN2-RGS9Mor ODN2-RGS2M. The group treated with ODN2-RGS2 received 30 nmolmorphine instead. All groups received, 24 h later, an i.c.v. test injection of10 nmol (or 30 nmol for ODN2-RGS2 group) morphine (A), and analgesiawas evaluated after 30 min. The time course of antinociception produced by10 nmol morphine was analysed in mice that had received saline or10 nmol morphine 24 h before. This was performed in mice undergoingODN2-RGS9M or ODN2-RGS9 treatment (B). Values are mean 6 SEMfrom groups of 10±20 mice. *P < 0.05 vs. the group injected with salinebefore the second dose of the opioid.

RGS9 facilitates opioid tachyphylaxis 805

ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 13, 801±811

609/EEC). Animals were lightly anaesthetized with ether and, to

reduce the possibility of interference from spinal events, all substances

were i.c.v.-injected as 4 mL volumes into the right lateral ventricle as

previously described (SaÂnchez-BlaÂzquez et al., 1995). The response of

the animals to nociceptive stimuli was determined in the warm water

(52 °C) tail-¯ick test. Baseline latencies ranged from 1.5 to 2.2 s.

Antinociception was expressed as a percentage of the maximum

possible effect, MPE = 100 3 (test latency ± baseline latency)/[cut-

off time(10 s) ± baseline latency]. For the different assays of the

study, either a ®xed dose or increasing doses of the opioid agonists

were given, and antinociception assessed at the interval of peak effect

[30 min after morphine and b-endorphin or, in time-course studies, at

various intervals after the injection of the opioids (Figs 2 and 3)]. In

chronic morphine assays, antinociception evoked by the opioid was

determined at several intervals after subcutaneous (s.c.) implantation

of the pellet. Statistical analysis of the results was performed by

analysis of variance (ANOVA) followed by the Student±Newman±

Keuls test (SigmaStat, SPSS Science Software, Erkrath, Germany).

The level of signi®cance was set at P < 0.05.

Evaluation of acute tolerance to morphine

A single i.c.v. injection of 10 nmol morphine was used to produce

acute tolerance. Controls were given saline instead. Analgesia was

evaluated 24 h later when the pretreatment had no effect on baseline

latencies. The opioid was i.c.v.-administered to these mice and acute

tolerance was determined by the decrease of antinociceptive potency.

Induction and assessment of tolerance upon chronic morphinetreatment

In one set of assays, the mice received a daily i.c.v. injection of

10 nmol morphine for four consecutive days, and the development of

tolerance was monitored by measuring the analgesic response

attained after each injection. In other assays, the animals were s.c.

implanted with 10 mL/kg body weight of a suspension containing

50% saline (0.9% NaCl in distilled water), 42,5% mineral oil (Sigma

#400±5), 7.5% Mannide Monooleate (Sigma #M-8546), and 0.1 g/

mL morphine base (adapted from SaÂnchez-BlaÂzquez et al., 1997).

Development of tolerance was determined by measuring the

analgesic response to the s.c.-implanted chronic opioid and to a

single i.c.v. dose of 10 nmol morphine that produced, in mice not

previously exposed to the opioid, an effect of » 80% MPE in the tail-

¯ick test.

Chemicals

(3-[125I]Iodotyrosyl27)-b-endorphin-(1-31) (human) (IM.162,

2000 Ci/mmol) was obtained from Amersham (Buckinghamshire,

England). Human b-endorphin (1±31) was purchased from

Peninsula Laboratories (San Carlos, CA, USA). Morphine sulphate

was acquired from Merck (Darmstadt, Germany), ICI-174,864

from CRB (Cambridge, UK), and leupeptin, aprotinin, bacitracin,

phenylmethylsulphonyl ¯uoride and BSA were purchased from

Sigma.

Results

Treatment with the ODNs selected for this study did not alter the

basal latencies of the animals in the tail-¯ick test. Base-line latencies

always ranged from 1.5 to 2.5 s at 52 °C. The impairment of RGS4,

RGS7, RGS9, RGS12, RGS14 or RGS16 brought about increases in

the amplitude and duration of opioid-evoked antinociception,

whereas knock down of RGS2 and RGS3 diminished these param-

eters (Fig. 1). The improvement of opioid activity was even more

pronounced when the antinociceptive activity declined, e.g. 90 min

after a single injection of morphine. The ODN to RGS5 did not

change the analgesic pro®le exhibited by this opioid. RGS9 and

RGS2 were selected to characterize these opposed actions on opioid

antinociception.

The ef®cacy and selectivity of the ODN treatments were assessed

with antibodies directed to RGS2 and RGS9 proteins. After resolving

mouse cerebral cortex, striatum and PAG by SDS-PAGE, the

antibodies recognized a protein of » 28 kDa for RGS2 (Heximer

et al., 1997) and of » 77 kDa for RGS9-2, respectively (Rahman et al.,

1999). In mice treated with the ODNs to RGS2 proteins, a signi®cant

decrease in the immunolabeling of antibodies to RGS2 was found. In

Fig. 1 the data corresponding to ODN2-RGS2 vs. ODN2-RGS2M are

presented. Percentage decreases were 55 6 5* for striatum, 44 6 4*

for cerebral cortex and 51 6 5* for PAG (Fig. 1). The three ODNs to

RGS9 proteins brought about similar decreases of RGS9-2 associated

immunoreactivity. Percentage decreases promoted by ODN2-RGS9

vs. ODN2-RGS9M on RGS9-2 immunosignals were: 40 6 4* for

striatum, 60 6 5* for cerebral cortex and 48 6 5* for PAG (values

are the mean 6 SEM from three independent experiments; values are

signi®cantly different from the ODN-mismatch group; P < 0.05,

ANOVA, Student±Newman±Keuls test). Treatment with the ODNs-

RGS2M, ODNs-RGS9M or the ODN-RD did not alter the immuno-

reactivity associated to RGS2 or RGS9-2 proteins. The ODNs directed

to RGS proteins distinct from RGS2 or RGS9 brought about no

changes in RGS2-or RGS9-2-associated immunoreactivity. The knock

down of RGS9-2 proteins did not alter the expression of other proteins

also implicated in opioid cellular signalling, e.g. Gb1/2 subunits, Gi2asubunits or the mu-opioid receptor (Fig. 1). Knock down of RGS2

proteins also lacked effect on these signalling elements (not shown).

FIG. 5. Lack of tolerance to morphine in RGS9 knock down mice.Following the standard schedule the mice received ODN2-RGS9 during the®rst 5 days. Controls received the ODN2-RGS9M. To maintain the knockdown of the RGS9 proteins for the study of the development of tolerance tomorphine, the ODN treatment continued with daily injection of 3 nmolODN for 5 more days. During this interval, and 2 h before the injection ofthe ODN, on days 7, 8, 9 and 10 the animals were i.c.v.-injected with10 nmol morphine, and antinociception determined at peak effect, 30 minlater, in the tail-¯ick test. Values are mean 6 SEM from groups of 10±20mice. *P < 0.05 vs. the group injected with morphine on day 7.

806 J. GarzoÂn et al.

ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 13, 801±811

In rat and human, the highest levels of RGS9-2 mRNA are found in

striatum and hypothalamus (Gold et al., 1997; Granneman et al.,

1998). In CD-1 mice, RT-PCR showed high signals for RGS9 mRNA

in striatum, but also low to medium levels in cerebral cortex and PAG

(Fig. 1).

In animals treated with the ODNs to RGS9, the potency of various

doses of morphine was signi®cantly enhanced over most of their

effective time course. The analgesia evoked by the opioid was

boosted and also became ef®cacious for longer postopioid intervals.

In using ODN2-RGS9, the apparent ED50s (i.c.v. morphine nmol/

mouse) and 95% con®dence limits in control mice were 3.51 (2.59±

4.71), 6.52 (4.67±9.03) and > 10 when determined 30, 60 and 90 min,

respectively, after the opioid. In RGS9 knock down mice, the

morphine ED50s at these intervals shifted to 1.10 (0.91±1.32), 2.31

(1.81±2.92) and 3.60 (2.53±5.11) nmol/mouse (Fig. 2). Similarly, in

animals treated with the ODNs to RGS9 proteins, the potency of

various doses of the opioid neuropeptide b-endorphin was signi®-

cantly enhanced over most of their effective time courses. Analgesia

was boosted and also made ef®cacious over longer postopioid

intervals. The ODNs to RGS2 proteins produced the opposite effects.

The activity of b-endorphin was diminished over the entire time

course. Results with ODN2-RGS2 and ODN2-RGS9 are shown in

Fig. 3. Impairment of RGS9 function brought about a leftward shift

of opioid dose±effect curves constructed at various postopioid

intervals, whereas de®cits of RGS2 proteins produced rightward

shifts (Figs 2 and 3).

The in¯uence of RGS2 and RGS9 on acute tolerance (tachyphy-

laxis) to morphine was also investigated. Morphine was used at

10 nmol/mouse in mice that had received ODN2 to RGS9, ODN2-

RGS9M and ODN2-RGS2M. Treatment with ODN2 to RGS2

produced marked decreases of morphine analgesic potency (Fig. 1).

Thus, in order to obtain an effect comparable to that produced by the

opioid in the other experimental groups, the dose of morphine was

raised to 30 nmol in ODN2-RGS2 mice. Mice received either saline

(control) or the opioid, and 24 h later the analgesia evoked by a second

injection of morphine was evaluated. In ODN2-RGS9M-treated mice,

antinociception reached 76 6 5% and 34 6 4% MPE (n = 12) in

control animals and in mice pretreated (±24 h) with the acute dose of

morphine. Morphine displayed no reduced activity when this assay

was performed in RGS9 knock down mice. Values of 72 6 5% and

74 6 4% MPE (n = 12) were recorded for controls and mice

pretreated with morphine, respectively (Fig. 4). In these mice, the

time course for morphine antinociception was not affected by the

previous dose of the opioid. The ODN-induced impairment of RGS2

proteins provided no protection against the acute tolerance evoked by

the single dose of 30 nmol morphine (Fig. 4).

To determine the in¯uence of RGS9 knock-down on the analgesic

effects evoked by daily injections of the opioid, the ODN treatment

was extended for 10 days. During the ®rst 5 days, treated mice

received the ODN2-RGS9 following the standard schedule, while

control animals received the ODN2-RGS9M. Afterwards, a daily

injection of 3 nmol ODN was given for 5 more days. The animals were

i.c.v.-injected with 10 nmol morphine on days 7, 8, 9 and 10. Control

mice exhibited a signi®cant decrease of morphine activity after the

second dose of the opioid given on day 8. No further decreases were

promoted by the morphine doses of days 9 and 10 (Fig. 5). RGS9

knock down mice exhibited normal responses to the consecutive doses

of morphine. Thus, acute tolerance to morphine as well as tolerance to

repeated injections of the opioid were greatly diminished in these

animals (Figs 4 and 5).

In the model of sustained morphine treatment, the impairment of

RGS9 proteins facilitated increases in the response to the delivered

opioid. This was only effective for 2±3 h after s.c.-implantation of the

morphine pellet. The opposite, a decrease in morphine activity, was

observed for RGS2 knock-down mice (Fig. 6A). In these mice, the

FIG. 6. In¯uence of RGS knock down on the development of tolerance tosustained chronic morphine. Animals received the ODNs into the rightlateral ventricle following a 5-day schedule. On day 6 the mice were s.c.-implanted at time zero with the oily suspension containing morphine.Development of tolerance was monitored by measuring the analgesiaproduced by the release of the s.c.-implanted opioid (A), and by a singlei.c.v. dose of 10 nmol morphine (B). This dose produced, in mice notpreviously exposed to the opioid, an effect of » 80% MPE in the tail-¯icktest. This is indicated in the ®gure with the dashed line. Values are themean 6 SEM from groups of 15±20 mice. *,+P < 0.05 vs. the group thathad received the mismatched control ODN-RGS9M or ODN-RGS2M,respectively, ANOVA, Student±Newman±Keuls test.

RGS9 facilitates opioid tachyphylaxis 807

ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 13, 801±811

increases or decreases in the potency of i.c.v. doses of morphine were

not evident 18±24 h after commencing chronic treatment with the

opioid (Fig. 6B). Thus, the impairment of RGS9 function did not bring

about long-term increases in the response of tolerant mice to i.c.v.

morphine.

Speci®c binding of 125I-Tyr27 human b-endorphin to mu-opioidreceptors in mouse PAG membranes

Membranes from mouse PAG were used in saturation assays of 125I-

Tyr27 human b-endorphin. Binding assays were performed in the

presence of 300 nM ICI-174,864 to prevent binding of labelled b-

endorphin to delta-opioid receptors. The data were analysed with the

LIGAND programme (Munson & Rodbard, 1980) and indicated a Kd

of » 0.3 nM and a binding capacity of » 50±60 fmol/mg of membrane

protein (present work; GarzoÂn et al., 1998). Thus, neither the af®nity of

the neuropeptide to mu-opioid receptors, nor its binding capacity,

changed due to treatment with ODNs directed towards RGS2 or RGS9

proteins (Fig. 7).

Discussion

The ODNs directed to RGS2 and RGS9 proteins effectively reduced

the expression of the respective proteins, but did not alter the

presence of other proteins required for signalling via the mu-opioid

receptors, i.e. Gi2a subunits, Gb1/2 subunits. Moreover, the number

of mu-opioid receptors and the af®nity displayed towards the agonists

were also unchanged. The use of ODNs revealed that RGS2 and

RGS9 proteins modulated opioid activity by a push±pull mechanism.

Signi®cant structural and functional differences exist between RGS9

and RGS2 which might account for RGS selectivity towards Gasubunits, and for the kinetics of their interaction. RGS6, 7, 9 and 11

contain G-protein g-subunit-like domains that mediate their associ-

ation with Gb5, but not with the other Gb subunits (Snow et al., 1998;

Hepler, 1999; Zhang & Simonds, 2000). Functionally active RGS9,

but not RGS2, forms a tight complex with the long spliced variant of

Gb5 (Makino et al., 1999). RGS2 is described as selectively binding

Gqa in brain membranes, but not other Ga subunits (Gi, Go, Gs,

G12/13), and completely inhibits Gq-directed activation of phospho-

lipase Cb1 (Heximer et al., 1997). This RGS2 is unique because it is

the only RGS protein that does not interact with Gi family members

and also because its expression is dynamically responsive to neuronal

activity (Ingi et al., 1998). It has been shown that RGS2 mRNA levels

increase in response to activation of different G-protein-coupled

receptors (Burchett et al., 1999; Song et al., 1999). In the rat, the

mRNA of RGS9-2 is common in striatum and hypothalamus but is

less frequent in other brain structures such as the neocortex (Gold

et al., 1997; Granneman et al., 1998). In this species, RGS9-1 mRNA

is abundant in retina but hardly detected in brain. The present results

from CD-1 mice indicate a greater presence of the long RGS9-2

protein in striatum than in cerebral cortex or PAG. The protein in

these structures could originat from the detected RGS9 mRNA and/or

be supplied by striatal or hippocampal projections (Saper, 1985;

Boylan et al., 1986; Hoover & Strick, 1993).

The classes of G-proteins regulated by opioid receptors in the

production of analgesia are acted upon by RGS9 and RGS2 proteins,

i.e. Gi, Gz and Gq proteins (GarzoÂn et al. 2000). In their opposite role

in opioid analgesia, RGS2 and RGS9 proteins might exhibit

differences in their binding to Ga-GTP and release from Ga-GDP

(rapid for RGS2 and slow for RGS9). By increasing the availability of

certain Gbg dimers, RGS proteins would also affect opioid-regulated

effects, e.g. Gbg direct binding to inwardly rectifying K+ channels

FIG. 7. Speci®c binding of 125I-Tyr27 human b-endorphin to mu-opioidreceptors in mouse PAG membranes. The Scatchard plots show theiodinated neuropetide's binding to PAG membranes from mice i.c.v.-injected with the ODNs to RGS2 or RGS9 proteins. Increasingconcentrations of 125I-Tyr27 human b-endorphin in the presence of 300 nM

ICI-174,864 (GarzoÂn et al., 1998) were incubated with approximately0.2 mg/mL ®nal protein concentration at 25 °C for 90 min. The Kd valueswere derived using the Ligand weighted nonlinear least squares regressionprogramme (Munson & Rodbard, 1980).

808 J. GarzoÂn et al.

ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 13, 801±811

and increasing their probability of opening (Chuang et al., 1998; Han

et al., 1999). An augmented availability of regulated G-proteins, or

prolonged activity of Ga-GTP on the effectors, would account for the

extended time course of the agonist effects.

Reports in literature, and also this work, show that tachyphylaxis is

produced by a single dose of opioids (Huidobro et al., 1976; Vaught

& Takemori, 1979; Porreca et al., 1987). This suggests that acute

tolerance initiates when opioid agonists promote the activation and

segregation of Ga-GTP monomers from Gbg dimers. Agonist-

activated receptors then switch to a nonsignalling state (silent

receptors) (Fig. 8) until they couple once more with trimeric G-

proteins. Therefore, to re-establish an adequate number of active

receptors, Ga-GDP subunits need to bind Gbg to reconstitute the

trimeric G-proteins. However, the desired restoration of G-proteins is

inef®cient. The reconstitution of trimeric G-proteins will mostly

depend on the speed at which Ga-GTP is converted to Ga-GDP by

the RGS proteins (Berman & Gilman, 1998). These GAPs bind to

active Ga-GTP subunits, competitively antagonizing the productive

interaction between effector and Ga-GTP (Tesmer et al., 1997), and

promoting the Ga-GDP form capable of binding to Gbg dimers

(Berman & Gilman, 1998; Hepler, 1999). Structural studies indicate

that Gbg and RGS do not bind simultaneously to the same Ga-GDP

(Tesmer et al., 1997) therefore Ga-GDP subunits must be released

from RGS proteins before they reconstitute the trimeric G-proteins.

Although RGS proteins bind with high af®nity to the transition state

of Ga-GTP, they also bind with lower af®nity to Ga-GTP or Ga-

GDP forms (Hunt et al., 1996; Hepler et al., 1997). Consequently,

while RGS proteins remain bound to Ga subunits, Gbg-Ga±GDP

association is delayed (BuÈnemann & Hosey, 1998; Hepler, 1999).

RGS proteins exhibiting this negative regulation of G-protein

signalling might determine the rate of desensitization of G-receptors

(Neil et al., 1997; Chuang et al., 1998; Tseng & Zhang, 1998;

DiverseÂ-Pierluissi et al., 1999; Potenza et al., 1999).

The ®nding that RGS9 knock down animals did not exhibit

tachyphylaxis after a single dose of the agonists indicates that

phosphorylation (inactivation) of Ga subunits, probably by PKC

(Strassheim & Malbon, 1994), is not greatly responsible for acute

tolerance to opioids. The reduction of RGS2 function did not prevent

acute tolerance to morphine which effectively developed in RGS2

knock down mice. Several hours after commencing chronic morphine

FIG. 8. Diagrammatic model describing theassigned roles of RGS2 and RGS9 on G-protein reconstitution. RGS2 proteins, afterbinding to activated Ga-GTP subunits,promote the rapid formation and release ofGa-GDP, thus making re-constituted trimericGabg proteins available to receptor regulation.Binding of RGS9 proteins to Ga-GTP delaysthe release of the Ga-GDP form much longer,allowing the signals originating at agonist-activated receptors to decline. Depletion ofRGS2 proteins decreases opioid potency.However, impairment of RGS9 proteins bringsabout marked enhancements of opioid potencyand the duration of its effects.

RGS9 facilitates opioid tachyphylaxis 809

ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 13, 801±811

treatment, the bene®cial effects of RGS9 knock down disappeared

and tolerance occurred. To reduce the impact of the chronic morphine

acting on the opioid receptors, other RGS proteins presumably

substitute the GAP function of RGS9 upon activated Ga-GTP

subunits.

These ®ndings indicate that over the time course of an opioid effect

there is a progressive reduction in the number of receptor-regulated

G-proteins. The agonist-induced separation of activated Ga-GTP

subunits from Gbg dimers is faster than the reconstitution of the

trimeric Gabg proteins. Hence tachyphylaxis develops and helps to

control the intensity of exogenous signals that could threaten cell

survival. Acute tolerance to opioids then arises as a bene®cial

membrane-delimited process caused by the RGS-facilitated progres-

sive depletion of receptor-regulated G-proteins. During this time, the

receptors remain capable of regulating new G-proteins. However,

long-term tolerance that occurs after repeated doses of opioids is

mostly due to uncoupling of opioid receptors from the regulated

transduction.

Acknowledgements

This work was supported by funding from the `ComisioÂn Interministerial deCiencia y TecnologõÂa' (grant CICYT SAF98-0057) and the `ProgramaSectorial de PromocioÂn General del Conocimiento' (grant DGICYT PB98-0649). Marta RodrõÂguez-DõÂaz is a predoctoral fellow from CAM. AlmudenaLoÂpez-Fando is a predoctoral fellow from FPI (MEC).

Abbreviations

GAP, GTPase-activating protein; G-protein, guanine nucleotide-bindingregulatory protein; ICI-174,864, N,N-diallyl-Tyr-(a-aminoisobutyric acid)2-Phe-Leu-OH; MPE, maximum possible effect; ODN, antisense oligodeoxy-nucleotide; ODN-RD, random ODN; ODN-RGS#M, mismatched antisensesequence; PAG, periaqueductal grey matter; RGS, regulator of G-proteinsignalling protein; TTBS, TBS-0.05% Tween 20.

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