Tolerance to Μ-Opioid Agonists in Human Neuroblastoma SH-SY5Y

Home , Naloxonazine

Tolerance to m-opioid agonists in human neuroblastoma SH-SY5Y cells as determined by changes in guanosine-5'-O-(3-[35S]-thio)triphosphate binding

Jackie Elliott, Li Guo & 1John R. Traynor

Department of Chemistry, Loughborough University, Leics., LE11 3TU and Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan, 48109, U.S.A.

1 The agonist action of morphine on membranes prepared from human neuroblastoma SH-SY5Y cells was measured by an increase in the binding of the GTP analogue [35S]-GTPgS. Morphine increased the 35 71 binding of [ S]-GTPgS to SH-SY5Y cell membranes by 30 fmol mg protein with an EC50 value of 76+10 nM. 2 Incubation of SH-SY5Y cells with 10 mM morphine for 48 h caused a tolerance to morphine manifested by a 2.5 fold shift to the right in the EC50 value with a 31+6% decrease in the maximum stimulation of [35 S]-GTPgS binding. The response caused by the partial agonist pentazocine was reduced to a greater extent. 3 Chronic treatment of the cells with the more ecacious m-ligand [D-Ala2, MePhe4, Gly-ol5]enkephalin (DAMGO, 10 mM) for 48 h aorded a greater eect than treatment with morphine. The maximal agonist eect of morphine was reduced to 58.9+6% of that seen in control cells while the maximal eect of DAMGO was reduced to 62.8+4%. There was a complete loss of agonist activity for pentazocine. 4 The development of tolerance was complete within 24 h and was blocked by naloxone and by the nonselective protein kinase inhibitor H7, but not by the putative b-adrenoceptor kinase (b-ARK) inhibitor suramin. 5 The observed tolerance eect was accompanied by a down-regulation of m-opioid receptors determined by a decrease in the maximal binding capacity for the opioid antagonist [3H]-diprenorphine of 66+4%, but with no change in binding anity. Binding of the agonist [3H]-DAMGO was similarly reduced. 6 The modulation of [35S]-GTPgS binding in SH-SY5Y cell membranes by opioids provides a simple method for the study of opioid tolerance at a site early in the signal transduction cascade. Keywords: m-Opioid receptor agonists; morphine; DAMGO; [35S]-GTPgS binding; tolerance; SH-SY5Y cells

Introduction m-Opioid receptors belong to the family of 7-transmembrane The agonist action of m-opioids can be functionally de- domain receptors which couple to heteromeric G proteins (Uhl monstrated in membranes from undierentiated SH-SY5Y et al., 1993). Continual exposure of opioid receptors to ago- cells by an increase in the level of binding of the guanosine 5'- nists leads to a tolerance caused by an initial desensitization of triphosphate (GTP) analogue [35S]-guanosine-5'-O-(g-thiotri- the receptor due to uncoupling, followed by down-regulation phosphate) ([35S]-GTPgS) (Traynor & Nahorski, 1995). This as the number of cell surface receptors decreases. The actual follows coupling of agonist-occupied receptors to Go and Gi molecular mechanisms governing these processes are unclear, proteins, as evidenced by the sensitivity of the system to per- though phosphorylation is believed to play an important role tussis toxin, and allows dierentiation of opioids of varying (Nestler, 1993). intrinsic activity. In this study changes induced in this coupling m-Opioid receptors are expressed, along with a lesser num- mechanism following chronic morphine treatment were asses- ber of d-opioid receptors, on SH-SY5Y human neuroblastoma sed as a reduction in the ability of opioid agonists to stimulate cells (Kazmi & Mishra, 1987; Yu & Sadee, 1988) and are ne- [35S]-GTPgS binding. The changes observed are a small (2- to 3 gatively coupled to adenylyl cyclase (Yu & Sadee 1988). Dif- fold) shift in potency accompanied by a reduction in the ferentiation of SH-SY5Y cells with retinoic acid leads to an up- maximal agonist-stimulated binding of [35S]-GTPgS, even with regulation of these receptors (Yu & Sadee, 1988). Chronic the highly ecacious agonist [D-Ala2, MePhe4, Gly-ol5]enke- treatment of such dierentiated cells with morphine leads to a phalin (DAMGO). The system provides a suitable method to decrease in the capacity of m-opioid agonists to inhibit the investigate mechanisms of tolerance occuring at the level of accumulation of adenosine 3' :5'-cyclic monophosphate (cyclic receptor-G protein. A preliminary account of part of this work AMP) (Yu & Sadee, 1988; Carter & Medzihradsky 1993a) and has been presented (Elliott & Traynor, 1994). to mediate inhibition of the N-type Ca2+ current (Kennedy & Henderson, 1991). These eects are accompanied by a decrease in the maximal binding capacity of m-ligands in cell membranes Methods (Zadina et al., 1994). Similar ®ndings have been obtained in undierentiated SH-SY5Y cells (Carter & Medzihradsky, Cell culture and membrane preparation 1992; Prather et al., 1994). Undierentiated human neuroblastoma SH-SY5Y cells (pas- sage number 78 ± 100) were cultured in Minimum Essential 1 Author for correspondence at: Department of Pharmacology, Medium supplemented with 10% foetal calf serum and anti- University of Michigan Medical School, 1301 Medical Research biotics, as described previously (Traynor & Nahorski, 1995). Building III, Ann Arbor, Michigan 48109-0632, U.S.A. Brie¯y, cells were grown in monolayers to con¯uency at 378C J. Elliott et al [35S]-GTPgS binding and opioid tolerance 1423 in a humidi®ed 5% CO2 atmosphere. The cells were harvested chem-Novabiochem (Nottingham, U.K.). Morphine sulphate in HEPES (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulpho- (MacFarlan Smith, Edinburgh, U.K.), suramin (Bayer plc, nic acid], 20 mM, pH 7.4)-buered saline containing 1 mM Newbury, U.K.) and TIPP (TyrTicPhePhe where Tic=te- EDTA, dispersed by agitation and collected by centrifugation trahydroisoquinoline-3-carboxylic acid; Dr. P. Schiller, at 5006g. For [35S]-GTPgS assays the cell pellet was suspended Clinical Research Institute, Montreal, Canada) were gifts. in a buer of 20 mM HEPES, pH 7.4, 100 mM NaCl and Minimal essential medium, foetal calf serum, fungizone, 10 mM MgCl2;6H2O (buer A) and homogenized with a tissue penicillin/streptomycin and L-glutamine were from GIBCO tearor (Biospec Products). The resultant homogenate was (Paisley, Scotland). Pertussis toxin and all other chemicals centrifuged at 50,000 6 g and the pellet collected, washed in were from Sigma and were of analytical grade. buer A and recentrifuged. The pellet was ®nally resuspended in buer A to give a protein concentration of between 100 ± 200 mgml71 (Lowry et al., 1951). All procedures were per- Results formed at 48C and membranes freshly prepared. In experiments to determine opioid binding in membranes, buer A was Morphine stimulated the binding of [35S]-GTPgS (80 pM)to replaced with 50 mM Tris-HCl buer, pH 7.4, in all procedures membranes from undierentiated SH-SY5Y cells in a con- and the ®nal pellet was resuspended to a protein concentration centration-dependent manner, as shown previously (Traynor 71 of approximately 100 mgml . To examine the eect of & Nahorski, 1995), aording an EC50 of 76.1+9.7 nM (n=9) chronic opioid exposure con¯uent cells were fed ®ve days after and a maximal stimulation of 30 fmol of [35S]-GTPgS passaging, and morphine (10 mM), DAMGO (10 mM) or sterile bound mg71 protein (Figure 1). The opioid partial agonist vehicle (water), was added to the medium. Two days later, pentazocine aorded a maximum level of stimulation of only unless otherwise stated, the cells were harvested and mem- 30% of that seen with morphine, with an EC50 of 129+51 nM branes prepared as above. Morphine (10 mM) or DAMGO (Figure 1). (10 mM) was added to control cells 5 min before harvesting to Pretreatment of undierentiated SH-SY5Y cells with 10 mM control for any eects due to residual opioid (Yu & Sadee, morphine for 48 h, conditions which have been shown to in- 1988; Zadina et al., 1993). duce down-regulation of m-opioid receptors in dierentiated SH-SY5Y cells (Zadina et al., 1994), caused a signi®cant 35 [ S]-GTPgS binding assays (P50.01) change in the morphine EC50 value to 190.7+36.8 nM (n=9) and signi®cantly (P50.01) reduced the SH-SY5Y cell membranes (100 ± 200 mg protein), prepared as maximal stimulation of [35S]-GTPgS binding by morphine to described above, were incubated in buer A containing [35S]- 69.3+6.3% (n=9) of control values (Figure 1). A similar re- GTPgS (80 pM), GDP (3 mM) and varying concentrations of duction (51.0+7.9% n=3) in the stimulation aorded by the opioid (1 ± 10,000 nM) in a total volume of 1 ml, for 60 min at partial agonist pentazocine was seen at the highest concen- 308C as described previously (Traynor & Nahorski, 1995). tration tested (Figure 1) and in the stimulation of [35S]-GTPgS Nonspeci®c binding was de®ned with unlabelled [35S]-GTPgS binding caused by a maximal concentration of the more e- (10 mM). Bound and free [35S]-GTPgS were separated by va- cacious fentanyl, which was reduced to 69.7+4.9% (n=14) of cuum ®ltration through GF/B ®lters and quanti®ed by liquid control values. Following pretreatment of SH-SY5Y cells for scintillation counting. EC50 values were determined by use of various times with morphine (10 mM) the loss of maximum Graphpad Prism, version 1.02 (GraphPad, San Diego, CA, stimulant eect of subsequently added morphine occurred over U.S.A.) and compared as log EC50 values by Student's paired t 24 h, with no further loss of eect when cells were exposed to test. morphine for up to 72 h (Table 1). When the cells were incubated with morphine (10 mM) for Opioid binding assays 48 h in the presence of naloxone (10 mM) there was no change

Cell membranes (100 mg of protein) prepared as described above were incubated in 50 mM Tris-HCl buer, pH 7.4, with varying concentrations (0.04 ± 10 nM) of the m-opioid agonist 15000 [3H]-DAMGO, or the nonselective antagonist [3H]-diprenorphine, in a total volume of 1 ml, for 40 min at 258C, as described previously (Traynor & Wood, 1989). Competition assays with [3H]-diprenorphine (0.48 nM) were performed at 10000 378C for 60 min in buer A containing GDP (3 mM), or, for whole cell assays, in Krebs/HEPES buer composition in mM: NaHCO3 25, NaCl 118, KCl 4.7, CaCl2.2H2O 2.6, KH2PO4 1.17, MgSO4.7H2O 1.18, glucose 11.7 and HEPES 10, pH 7.4 for 60 min at 37 C. In all opioid binding experiments non- 8 5000 speci®c binding was de®ned with naloxone (10 mM), and bound and free ligand were separated by vacuum ®ltration and Stimulation (d.p.m.) quanti®ed by liquid scintillation counting. Total numbers of binding sites (Bmax) and anities (Kd) were determined by the program LIGAND (Munsen & Rodbard, 1980) and anities from competition assays (Ki) calculated with the Cheng and 1 10 100 1000 10000 Prusso equation (Cheng & Prusso, 1973). Agonist (nM)

Chemicals and drugs Figure 1 Stimulation of [35S]-GTPgS binding by morphine (circles) and pentazocine (triangles) in membranes from control (solid [35S]GTPgS (guanosine-5'-O-(3-[35S]-thiotriphosphate 46.1- symbols) or morphine (10 mM, 48 h)-pretreated (open symbols) SH- 51.5 TBq mmol71) was from New England Nuclear (Steve- SY5Y cells. The response to morphine in cells pretreated with 3 3 2 4 morphine (10 M, 48 h) in the presence of naloxone (10 M)is nage, U.K.) and [ H]-DAMGO ([ H]-[D-Ala ,MePhe ,Gly- m m represented by the squares. Incubations were performed at 30 C for ol5]enkephalin 1.49 TBq mmol71) and [3H]-diprenorphine 8 71 1 h as described in Methods. Values represent means and vertical (1.78 TBq mmol ) from Amersham International (Ayles- lines show s.e. from at least three separate experiments (nine separate bury, U.K.). Unlabelled DAMGO, naloxone and pentazo- experiments for morphine) performed in duplicate. Maximal cine were purchased from Sigma (Poole, U.K.) and H7 (1- stimulation by morphine was equivalent to 30.0+1.7 fmol [35S]- (5-isoquinolinesulphonyl)-2-methylpiperazine) from Calbio- GTPgS bound mg71 protein. 1424 J. Elliott et al [35S]-GTPgS binding and opioid tolerance

Table 1 Stimulating eects of morphine on the binding of 35 a [ S]-GTPgS to membranes prepared from SH-SY5Y cells exposed to morphine (10mM) for varying times 100

Time of exposure Maximal eect EC50 (h) (%) (nM) 80 0* 100 76.1+9.7 1.5 96.3+2.4 134+42 3 87.6+4.9 131+30 6 77.8+6.2 140+35 60 12 83.7+3.7 101+26 24 72.8+6.8 129+19 48* 69.3+6.3** 191+37** 40 72 70.4+1.2 157+14

SH-SY5Y cells were incubated with morphine (10 mM) for % fentanyl maximum the stated times. Membranes from these cells were prepared 20 as in the Methods section and incubated with [35S]-GTPgS (100 pM) and GDP (3 mM) for 60 min at 308C, with increasing concentrations of morphine. Maximal eect and EC50 values were determined from concentration-eect 10 100 1000 10000 curves of the data. Values are means+s.e. from three (or Morphine (nM) *nine) experiments performed in triplicate. (**P50.01, compared to control cells).

b 100 in the morphine-induced stimulation of [35S]-GTPgS binding compared to control, naive, cells (Figure 1). However, naloxone itself had a small eect on the basal level of [35S]-GTP S g 80 binding in both control and morphine-treated cells. In control cells naloxone (10 mM) stimulated binding of [35S]-GTPgSby 3.0 0.4 fmol [35S]-GTP S bound mg71 protein but in mor- + g 60 phine pretreated cells inhibited binding by 2.4+0.1 fmol [35S]- GTPgS bound mg71 protein (n=3). This compares with the increase in binding induced by morphine (10 M) in control m 40 cells of 30.0+1.7 fmol [35S]-GTPgS bound mg71 protein and in pretreated cells of 20.2+3.9 fmol [35S]-GTPgS bound mg71 protein. The inhibitory eect of naloxone was not due to re- % fentanyl maximum 20 sidual morphine since this was added to control cells imme- diately before the assay. Exposure of SH-SY5Y cells to the highly ecacious m- agonist, DAMGO (10 mM), for 48 h caused a greater loss of 10 100 1000 10000 responsiveness of [35S]-GTPgS binding compared to morphine (10 mM)-pretreated cells. Thus the maximal morphine-medi- DAMGO (nM) ated stimulation of [35S]-GTPgS binding was reduced to 58.6+6.6% (P50.01) of the level seen in naive cells (Figure c 2a), with a shift in the EC50 to 341+68.5 nM (P50.05). Chronic DAMGO treatment of the cells also reduced the ef- 100 fectiveness of DAMGO itself to stimulate [35S]-GTPgS binding, reducing the maximum eect to 62.8+3.95% (P50.01) of the response in control cells and aording EC values of 50 80 46.2+8.1 nM and 240+50.4 nM (P50.01) in membranes from naive and tolerant cells, respectively (Figure 2b). The maximal response to fentanyl was reduced to 42.5+5.9% (n=12) of the control level. The increased eectiveness of chronic DAMGO 60 over chronic morphine pretreatment was particularly marked for the partial agonist pentazocine, where a complete loss of the stimulant eect was observed (Figure 2c). Chronic incu- 40 bation with morphine or DAMGO had no eect on the basal 35 level of [ S]-GTPgS binding. % fentanyl maximum Incubation of SH-SY5Y cells with the non-speci®c protein 20 kinase inhibitor H7 (50 mM) (Hidaka et al., 1984), during chronic exposure to morphine (10 mM, 48 h), largely prevented the reduction in agonist modulation of [35S]-GTPgS binding (Figure 3). H7 treatment alone did not alter the maximum level 10 100 1000 10000 35 of stimulation of [ S]-GTPgS binding over basal binding Pentazocine (nM) aorded by DAMGO, morphine or pentazocine, nor did acute 35 H7 have any eect on the response of the [35S]-GTPgS binding Figure 2 Concentration-eect curves for the stimulation of [ S]- to morphine. However, H7 has been shown to compete GTPgS binding by (a) morphine, (b) DAMGO and (c) pentazocine to membranes from naive SH-SY5Y cells (solid symbols) and cells directly with opioid receptor binding (Aloyo, 1995). To deter- pretreated for 48 h with DAMGO (10 mM) (open symbols). Values mine if H7 was acting by interfering with the binding of mor- are given as a percentage of the maximal response to fentanyl in phine to m-opioid receptors in whole SH-SY5Y cells naive cells. Assays were conducted at 308C for l h and values competition experiments against [3H]-diprenorphine in Krebs- represent means with vertical lines showing s.e. from at least three HEPES buer were performed. However, the anity (Ki)ofH7 separate experiments performed in duplicate. J. Elliott et al [35S]-GTPgS binding and opioid tolerance 1425 for the m-opioid receptor was 4500 mM compared to the Ki for maximal stimulation induced by pentazocine was less, con- morphine, determined under the same conditions, of ®rming the partial agonist nature of this opioid. These obser- 566+38 nM (Hill coecient of 0.89+0.10). In contrast to H7, vations are similar to those previously obtained (Traynor & incubation of SH-SY5Y cells with the putative b-adrenoceptor Nahorski, 1995) but the compounds were less potent, for ex- kinase (b-ARK) inhibitor suramin (Miller et al., 1993) (10 mM) ample the EC50 value for morphine was 2.7 fold lower and for during exposure of the cells to morphine (10 mM for 48 h), did pentazocine was 1.4 fold lower. However, binding assays with not prevent the loss in m-agonist-modulation of [35S]-GTPgS the selective m-opioid agonist [3H]-DAMGO revealed a lower binding by either morphine, DAMGO or pentazocine (Figure number of opioid m-receptors in the SH-SY5Y cells used in 3). these studies (140 fmol mg71 protein) compared to previous The undierentiated SH-SY5Y cells used in this study studies (254 fmol mg71 protein). The shift to lower potency expressed m-opioid receptors at a level (Bmax) of 140+ with a reduction in receptor number agrees with accepted 11 fmol mg71 protein, as measured with [3H]-DAMGO, with theories of receptor occupancy (Kenakin, 1993) and con®rms 3 an anity (Kd) for [ H]-DAMGO of 1.45+0.31 nM. Chronic the usefulness of the system for investigating pharmacological DAMGO treatment (10 mM, 48 h) of the cells caused a re- concepts. The tritiated antagonist [3H]-diprenorphine did af- 3 71 71 duction in the Bmax for [ H]-DAMGO to 71+7 fmol mg ford a maximal number of binding sites (206 fmol mg pro- protein, but no signi®cant change in the measured anity (Kd, tein) higher than the number of sites labelled by the m-agonist 0.88+0.18 nM). By use of the antagonist [3H]-diprenorphine to [3H]-DAMGO. This suggests that the antagonist [3H]-dipre- label the opioid sites the number of binding sites in naive cells was determined as 206+36 fmol mg71 protein, and dropped to 65+14 fmol mg71 protein in DAMGO-pretreated cells (Figure 4). The ability of the agonist DAMGO to compete for [3H]-diprenorphine (0.5 nM) binding sites in SH-SY5Y cell membranes in buer A, as used for [35S]-GTPgS assays, was similar in membranes from both control and morphine-treated 1500 60 (10 M, 48 h) cells aording K values of 79 12 and m i + 40 60.3+27 nM, respectively (Figure 5). These Ki values are high 20 when compared to expected values obtained in low ionic Bound/free strength buers (e.g. Tris) since the assays were performed 1000 35 under the conditions of the [ S]-GTPgS binding assay, which 0 10 20 promotes the formation of lower anity states of the -re- m Bound (fmol/assay) ceptor (Carroll et al., 1988; Traynor & Nahorski, 1995). The selective -ligand TIPP at a concentration of 10 nM, that is 30 d 500 times its anity for d-receptors (Schiller et al., 1992), displaced Specific binding (d.p.m.) only 19.8+1.5% (n=3) of speci®cally bound [3H]-diprenorphine (0.5 nM).

Discussion 0 1 2 3 4 [3H]-diprenorphine (nM)

Binding the m-opioid agonists morphine and DAMGO to 3 membranes from undierentiated SH-SY5Y cells caused an Figure 4 Saturation binding of [ H]-diprenorphine to control (*) increase in the binding of the GTP analogue [35S]-GTP S. The and DAMGO-treated (10 mM, 48 h) (O) SH-SY5Y cells. Binding g assays were performed in SH-SY5Y cell membranes in Tris buer (pH 7.4, 50 mM) for 40 min at 258C as described in Methods. This is a representative experiment which was replicated three times. Inset * shows the Scatchard transformation of the data. 100 * * 100

60 75 % control 40

% control specific binding 20

50 Morphine DAMGO Pentazocine 0 Figure 3 Eect of a single concentration (3 mM) for each of the 0.1 1 10 100 1000 10000 agonists morphine, DAMGO and pentazocine on the stimulation of DAMGO (nM) [35S]-GTPgS binding to membranes of SH-SY5Y cells pretreated for 3 48 h with morphine (10 mM) alone (open columns) or in the presence Figure 5 Displacement of the speci®c binding of [ H]-diprenorphine of H7 (50 mM; hatched columns) or suramin (10 mM; stippled (0.49 nM) by DAMGO in control (*) and morphine-treated (10 mM, columns). Values are given as a percentage of the maximal response 24 h) (O) SH-SY5Y membranes, conducted in buer A (20 mM to each agonist in naive cells. Assays were conducted at 308C for 1 h HEPES, 100 mM NaC1 and 10 mM MgC12.6H2O, pH 7.4) in the and values represent means+s.e. from at least three separate presence of 3 mM GDP at 308C for 1 h as described in Methods. experiments performed in duplicate. *Not signi®cantly dierent from Values represent means with vertical lines showing s.e. from at least control values (P50.05, Student's t test). three separate experiments performed in duplicate. 1426 J. Elliott et al [35S]-GTPgS binding and opioid tolerance norphine labels both high and low anity states of the opioid for morphine but also for the highly ecacious agonists m-receptor, whereas [3H]-DAMGO will only bind to m-opioid DAMGO and fentanyl. Although unexpected, similar ®ndings receptors in a high anity state (Carroll et al., 1988; Traynor have been obtained. Thus, in retinoic acid-dierentiated SH- & Wood, 1989). However, there is a contribution to the ob- SY5Y cells chronically treated with DAMGO, a decreased served [3H]-diprenorphine binding from a d-opioid receptor maximal eect of DAMGO inhibition of adenylyl cyclase and population since the selective d-antagonist TIPP (Schiller et al., stimulation of GTPase is seen (Carter & Medzihradsky, 1992) blocked 20% of the speci®c binding of [3H]-diprenor- 1993a). However, this eect is not seen with chronic morphine phine, and others have suggested the presence of k3 binding pretreatment. On the other hand, chronic exposure of 7315c sites on these cells (Cheng et al., 1995). pituitary cells to morphine for 3 h results in a reduction in the Incubation of the SH-SY5Y cells with morphine (10 mM)or maximal ability of the full-agonist [D-Ala2, D-Leu5]enkephalin DAMGO (10 mM) for 48 h led to a decrease in the ability of to reduce adenylyl cyclase activity and, with longer exposure to subsequently added m-opioid agonists to stimulate the binding morphine, a complete loss of the activity (Puttfarcken et al., of [35S]-GTPgS to membranes of SH-SY5Y cells. The eect of 1988). opioid pretreatment was manifest by a rightward shift in the The ecacy of drugs is not only related to the drug but to 35 EC50 value and a reduction in the maximal [ S]-GTPgS bind- tissue factors. For example, morphine is a full agonist in the ing, even for the ecacious m-opioid DAMGO. This was most guinea-pig ileum, a partial agonist in the mouse vas deferens, marked for the partial agonist pentazocine which completely but an antagonist in the rat vas deferens (Traynor, 1994). lost the ability to stimulate [35S]-GTPgS binding in membranes These eects can be explained by dierences in receptor re- from cells which had been treated chronically with DAMGO. serve. A low m-opioid receptor reserve in SH-SY5Y cells, as Both the loss of maximal eect and the alteration in EC50 were observed for the cloned m-receptor expressed in CHO or BHK speci®c opioid-receptor mediated events, as indicated by the cells (Pak et al., 1996), could explain the present ®ndings. sensitivity of the processes to naloxone. Chronic treatment with However, there is evidence for a m-receptor reserve in SH- the more ecacious DAMGO aorded a greater eect than SY5Y cells since the ratio of Ki to EC50 values for stimulation that induced by chronic morphine treatment. of [35S]-GTPgS binding is 41 (Traynor & Nahorski, 1995). In The loss of agonist activity following chronic exposure of addition, at least in retinoic acid treated cells, reduction in the cells to morphine or DAMGO was accompanied by a re- receptor number by 50% with the alkylating agent b-chlor- duction in the number of m-opioid binding sites measured with naltexamine has no eect on the maximal inhibition of ade- either [3H]-DAMGO or [3H]-diprenorphine. A m-opioid re- nylyl cyclase and a 90% reduction in receptor number ceptor antagonist such as [3H]-diprenorphine which labels both following chronic treatment with levorphanol only reduces the coupled and uncoupled states of the m-receptor measures total maximal response by a small amount (Carter & Medzihradsky, receptor numbers, and con®rms an actual loss in membrane- 1993a). Other tissue factors altered by the tolerance process bound receptor number, as shown in both undierentiated may therefore be responsible for the reduction in maximal (Carter & Medzihradsky, 1992; Prather et al., 1994) and reti- [35S]-GTPgS binding. It is known that the m-opioid receptor in noic acid-dierentiated SH-SY5Y cells (Zadina et al., 1994), undierentiated SH-SY5Y cells activates several dierent, rather than just a desensitization and uncoupling from G pertussis-toxin sensitive, heterotrimeric G proteins (Laugwitz proteins. The loss of receptor number decreased to a plateau et al., 1993). The [35S]-GTPgS binding assay does not distin- level at approximately 70 fmol mg71 protein, measured with guish these dierent G proteins but rather represents a mea- either [3H]-DAMGO or [3H]-diprenorphine, suggesting that sure of all G proteins activated following agonist occupation of approximately 40% of the m-opioid receptors are refractory to a particular receptor. The reduced maximal response to the full down-regulation. These `refractory' receptors are con®rmed to agonist DAMGO may indicate that the loss of receptors is be of the m-type, as indicated by the anity of DAMGO (Kd accompanied by a reduction in associated G proteins or altered 0.9 nM) determined from the saturation binding curve. This function of these G proteins, such that there is a reduced ca- may indicate the presence of two pools of m-opioid receptors, pacity for [35S]-GTPgS binding. Indeed, chronic treatment of only one of which is sensitive to chronic opioid treatment. rat striatal neurones with morphine causes a reduction in the However, both pools appear to have very similar properties number of inhibitory G-proteins (Van Vliet et al., 1993) and since m-opioid receptors in naive and morphine pretreated cells chronic DAMGO treatment of retinoic acid dierentiated SH- were not distinguishable in saturation binding assays or in the SY5Y cells decreases the content of Goa, the G protein re- displacement of [3H]-diprenorphine binding by DAMGO and sponsible for coupling opioid m-receptors to adenylyl cyclase in 35 the EC50 values for stimulation of [ S]-GTPgS binding diered these cells (Carter & Medzihradsky, 1993b). However, any only by 2 ± 3 fold. It will be intriguing to discover if the m- such G protein changes would have to be speci®c for the m- opioid binding site population resistant to down-regulation opioid receptor since in SH-SY5Y cells tolerance to m-opioid relates to the ®nding that the eects of opioids on pupil size agonists appears to be homologous (Kennedy & Henderson, and the constipating eects of opioids are generally resistant to 1991; Lameh et al., 1992; Prather et al., 1994). tolerance (Adams & Holtzman, 1990). In naive SH-SY5Y cells naloxone showed a small degree of A reduction in receptor number, as observed following activity resulting in stimulation of [35S]-GTPgS of 10% of that morphine or DAMGO pretreatment of the cells, would be seen with morphine. In contrast, in morphine pre-treated cells expected to result in a shift to the right in the concentration- naloxone caused a small inhibition of basal [35S]-GTPgS eect curve for a full agonist, accompanied by a reduction in binding, indicating a possible inverse agonist action. These the maximal eect for a less ecacious compound (Kenakin, results agree with ®ndings at the b2 receptor where, following 1993). In the locus coeruleus m-opioid receptors couple to an desensitization, very weak agonists may show inverse agonist inwardly rectifying K+ channel. Chronic treatment of rats with activity (Chidiac et al., 1996). Such an inverse agonist action of morphine results in a shift in the concentration curve for naloxone would lead to a reduction in the level of activated G DAMGO acting on this channel, while for the less ecacious protein and could be responsible for the overshoot in cyclic normorphine a ¯attening of the dose-eect curve occurs AMP production seen with naloxone following chronic treat- (Christie et al., 1987). Similarly, chronic morphine treatment of ment of SH-SY5Y cells with morphine (Wang et al., 1994). retinoic acid dierentiated SH-SY5Y cells leads to a 7 fold Opioid receptors may be substrates for b-adrenoceptor ki- shift in the ability of DAMGO to inhibit an N-type Ca2+ nase (b-ARK). Indeed, k-receptors in cells with dominant ne- current in these cells, whereas morphine shows a reduction in gative mutations of b-ARK do not down-regulate or the maximal eect (Kennedy & Henderson, 1991), and there is desensitize (Raynor et al., 1994). The present experiments have a reduction in the maximal inhibition of adenylyl cyclase by shown that the putative b-ARK inhibitor suramin at 10 mM, morphine (Yu & Sadee, 1988). In contrast, the present ®ndings that is 100 times higher than its reported activity in A431 cells in undierentiated SH-SY5Y cells show both a shift in the (Miller et al., 1993), did not block the development of tolerance 35 EC50 value and a decrease in the maximal response, not only as determined by changes in [ S]-GTPgS binding. In contrast, J. Elliott et al [35S]-GTPgS binding and opioid tolerance 1427 the non-speci®c protein kinase inhibitor H7 (Hidaka et al., do indicate that H7 can be used to study phosphorylation 1984) was able to prevent the eects of chronic morphine pre- processes associated with opioid receptor systems. treatment, supporting evidence that phosphorylation is a key In conclusion, the results show that the stimulation of [35S]- step in receptor desensitization and the development of toler- GTPgS binding mediated by m-opioid receptors in SH-SY5Y ance to opioids (Childers, 1991; Nestler, 1993; Raynor et al., cells is sensitive to long-term exposure to m-agonists. A toler- 1994; Wang et al., 1994; Pei et al., 1995; Ueda et al., 1995). It is ance develops that is manifested by a small reduction in ago- possible that the observed eects of H7 are unrelated to an nist potency together with a reduced maximal eect, even for a inhibition of kinase enzymes, but are due to a direct action of highly ecacious agonist such as DAMGO. The mechanism of the compound at opioid receptors (Aloyo, 1995). However, H7 this tolerance involves a reduction in receptor number and may displaced [3H]-diprenorphine speci®cally bound to whole cells require altered function of inhibitory G protein. This appears under physiological conditions (Krebs buer) with a Ki of to involve phosphorylation of components within the receptor 4500 mM, whilst the Ki for morphine was 566 nM, similar to and/or G protein systems. Since guanine nucleotide exchange values previously obtained in SH-SY5Y cells (Elliott et al., is the ®rst measurable biochemical event following agonist 1994) and guinea-pig brain membranes (Carroll et al., 1988). At occupation of seven-transmembrane domain G protein-linked the level of morphine (10 mM) used for the chronic studies it was receptors, the binding of [35S]-GTPgS can be used as a func- determined, by use of the Cheng and Prusso equation (Cheng tional measure to probe readily mechanisms behind the de- & Prusso, 1973), that approximately 5 mM H7 would be re- velopment of tolerance very early in the signal-transduction quired to displace 50% of the morphine from opioid receptors. cascade. It is unlikely, therefore, that H7, at the concentration used, prevented the eects of chronic morphine treatment by direct competition at m-opioid binding sites. Thus, unlike previous ®ndings in non-physiological buers (Aloyo, 1995), the results We thank the Wellcome Trust for ®nancial support.

References

ADAMS, J. & HOLTZMAN, S.R. (1990). Tolerance and dependence KENNEDY, C. & HENDERSON, G. (1991). m-Opioid receptor after continuous morphine infusion from osmotic pumps inhibition of calcium current: development of homologous measured by operant responding in rats. Psychopharmacology, tolerance in single SH-SY5Y cells after chronic exposure to 100, 451 ± 458. morphine in vitro. Mol. Pharmacol., 40, 1000 ± 1005. ALOYO, V.J. (1995). Modulation of mu opioid binding by protein KENAKIN, T. (1993). Pharmacologic Analysis of Drug-Receptor kinase inhibitors. Biochem. Pharmacol. 49, 17 ± 21. Interactions. 2nd edition, pp 249 ± 277. New York: Raven Press. CARROLL, J.A., SHAW, J.S. & WICKENDEN, A.D. (1988). The LAMEH,J.,EIGER,S.&SADEE,W.(1992). Interaction among m- physiological relevance of low agonist anity binding at opioid opioid receptors and a2-adrenoceptors on SH-SY5Y human m-receptors. Br. J. Pharmacol., 94, 625 ± 631. neuroblastoma cells. Eur. J. Pharmacol., 227, 19 ± 24. CARTER, B.D. & MEDZIHRADSKY, F. (1992). Opioid signal LAUGWITZ, K.-H., OFFERMANNS, S., SPICHER, K. & SCHULTZ, G. transduction in intact and fragmented SH-SY5Y neural cells. J. (1993). m and d opioid receptors dierentially couple to G protein Neurochem., 58, 1611 ± 1619. subtypes in membranes of human neuroblastoma SH-SY5Y cells. CARTER, B.D. & MEDZIHRADSKY, F. (1993a). Receptor mechan- Neuron, 10, 233 ± 242. isms of opioid tolerance in SH-SY5Y human neural cells. Mol. LOWRY, O.H., ROSENBERG, N.G., FARR, A.L. & RANDALL, R.J. Pharmacol., 43, 465 ± 473. (1951). Protein measurement with the Folin phenol reagent. J. CARTER, B.D. & MEDZIHRADSKY, F. (1993b). Go mediates the Biol. Chem., 139, 265 ± 275. coupling of m opioid receptor adenylyl cyclase in cloned neural MILLER, S.W., HASKE, T.N., RABY, C.A. & LEVINE, H. (1993). cells and brain. Proc. Natl. Acad. Sci. U.S.A., 90, 4062 ± 4066. Inhibition of b2 adrenergic receptor desensitization. Soc. CHENG, J., STANDIFER, K.M., TUBLIN, P.R., SU, W. & PASTERNAK, Neurosci Abs., 19, 181.5. G.W. (1995). Demonstration of k3 opioid receptors in the SH- MUNSON, P.J. & RODBARD, D. (1980). LIGAND: a versatile SY5Y human neuroblastoma cell line. J. Neurochem., 65, 170 ± computerised approach for characterisation of ligand-binding 175. systems. Anal. Biochem., 107, 220 ± 239. CHENG, Y.C. & PRUSSOFF, W.H. (1973). Relationship between the NESTLER, E.J. (1993). Cellular responses to chronic treatment with inhibition constant (Ki) and the concentration of inhibitor which drugs of abuse. Critical Rev. Neurobiol., 7, 23 ± 39. causes 50 per cent inhibition (I50) of an enzymic reaction. PAK, Y., KOUVELAS, A., SCHEIDELER, M.A., RASMUSSEN, J., Biochem. Pharmacol., 22, 3099 ± 3108. O'DOWD, B.F. & GEORGE, S.R. (1996). Agonist-induced func- CHIDIAC, P., NOUET, S. & BOUVIER, M. (1996). Agonist-induced tional desensitization of the m-opioid receptor is mediated by loss modulation of inverse agonist ecacy at the b2-adrenergic of membrane receptors rather than uncoupling from G protein. receptor. Mol. Pharmacol., 50, 662 ± 669. Mol. Pharmacol. 50, 1214 ± 1222. CHILDERS, S.R. (1991). Opioid-receptor coupled 2nd messenger PEI, G., KEIFFER, B.L., LEFKOWITZ, R.J. & FREEDMAN, N.J. (1995). systems. Life Sci., 48, 1991 ± 2002. Agonist-dependent phosphorylation of the mouse d-opioid CHRISTIE, M.J., WILLIAMS, J.T. & NORTH, R.A. (1987). Cellular receptor: involvement of G-protein-coupled receptor kinases mechanisms of opioid tolerance: studies in single brain neurons. but not protein kinase C. Mol. Pharmacol., 48, 173 ± 177. Mol. Pharmacol., 32, 633 ± 638. PRATHER, P.L., TSAI, A.W., & LAW, P.Y. (1994). Mu and delta opioid ELLIOTT, J., LAMBERT, D.G., SMART, D. & TRAYNOR, J.R. (1994). receptor desensitization in undierentiated human neuroblasto- Characterisation of m-opioid receptors on SH-SY5Y cells using ma cell SH-SY5Y cells. J. Pharmacol. Exp. Ther., 270, 177 ± 184. naloxonazine and b-funaltrexamine. Eur. J. Pharmacol. Mol. PUTTFARCKEN, P.S., WERLING, L.L. & COX, B.M. (1988). Eects Pharmacol., 268, 447 ± 450. of chronic morphine exposure on opioid inhibition of adenylyl ELLIOTT, J. & TRAYNOR, J.R. (1994). [35S]GTPgS binding in SH- cyclase in 7315c cell membranes: a useful model for the study SY5Y human neuroblastoma cells as a model for the study of of tolerance at m opioid receptors. Mol. Pharmacol., 33, 520 ± opioid tolerance. Regul. Peptides, 54, 91 ± 92. 527. HIDAKA, H., INAGAKI, M., KAWAMOTO, S. & SASAKI, Y. (1984). RAYNOR, K., KONG, H., HINES, J., KONG, G., BENOVIC, J., Isoquinolinesulfonamides, novel and potent inhibitors of cyclic YASUDA, K., BELL, G. & REISINE, T. (1994). Molecular nucleotide dependent kinase and protein kinase C. Biochemistry, mechanisms of agonist-induced desensitization of the cloned 23, 5036 ± 5041. mouse kappa opioid receptor. J. Pharmacol. Exp. Ther., 270, KAZMI, S.M. & MISHRA, R.K. (1987). Comparative pharmacological 1381 ± 1386. properties and functional coupling of m and d opioid receptor sites in human neuroblastoma SH-SY5Y cells. Mol. Pharmacol., 32, 109 ± 118. 1428 J. Elliott et al [35S]-GTPgS binding and opioid tolerance

SCHILLER, P.W., NGUYEN, T.M.D., WELTOWSKA, G., WILKES, B.C., VAN VLIET, B., VAN RIJSWIJK, A.L.C.T., WARDEH, G., MULDER, MARSDEN, B.C., LEMIEUX, C. & CHUNG, N.N. (1992). Dier- A.H. & SCHOFFELMEER, A.N.M. (1993). Adaptive changes in the ential stereochemical requirements of m versus d opioid receptors number of Gs- and Gi-proteins underlie adenylyl cyclase for ligand binding and signal transduction: development of a sensitization in morphine-treated rat striatal neurons. Eur. J. class of potent and highly d-selective peptide antagonists. Proc. Pharmacol., 245, 23 ± 29. Natl. Acad. Sci. U.S.A., 89, 11871 ± 11875. WANG, Z., BILSKY, E.J., PORRECA, F. & SADEE, W. (1994). TRAYNOR, J.R. (1994). Opioid receptors and their subtypes: Focus Constitutive m opioid receptor activation as a regulatory on peripheral isolated tissue preparations. Neurochem. Int., 24, mechanisms underlying narcotic tolerance and dependence. Life 427 ± 432. Sci., 54, 339 ± 350. TRAYNOR, J.R. & NAHORSKI, S.R. (1995). Modulation by m-opioid YU,V.C.&SADEE,W.(1988). Ecacy and tolerance of narcotic agonists of guanosine-5'-O-(3-[35S]thio)triphosphate binding to analgesics at the mu opioid receptor in dierentiated human membranes from human neuroblastoma SH-SY5Y cells. Mol. neuroblastoma cells. J. Pharmacol. Exp. Ther., 245, 350 ± 355. Pharmacol., 47, 848 ± 854. ZADINA, J.E., CHANG, S.L., GE, L.-J. & KASTIN, A.J. (1993). Mu TRAYNOR, J.R. & WOOD, M.S. (1989). [3H]Diprenorphine binding to opiate receptor down-regulation by morphine and up-regulation kappa-sites in guinea-pig and rat brain: evidence for apparent by naloxone in SH-SY5Y human neuroblastoma cells. J. heterogeneity. J. Neurochem., 53, 173 ± 178. Pharmacol. Exp. Ther., 265, 254 ± 262. UEDA, H., MIYAMEA, T., HAYASHI, C., WATANABE, S., FUKUSH- ZADINA, J.E., HARRISON, L.M., GE, L.-J., KASTIN, A.J. & CHANG, IMA, N., SASAKI, Y., IWAMURA, T. & MISU, Y. (1995). Protein S.L. (1994). Dierential regulation of mu and delta opiate kinase C involvement in homologous desensitization of the d- receptors by morphine, selective agonists and antagonists and opioid receptor coupled to Gi1-phospholipase C activation in dierentiating agents in SH-SY5Y human neuroblastoma cells. J. xenopus oocytes. J. Neurosci., 15, 7485 ± 7499. Pharmacol. Exp. Ther., 270, 1086 ± 1096. UHL, G.R., CHILDERS, S. & PASTERNAK, G.W. (1993). An opiate- receptor gene family reunion. Trends Pharmacol. Sci., 17, 89 ± 93. (Received December 12, 1996 Revised April 7, 1997 Accepted April 16, 1997)