Proc. NatL. Acad. Sci. USA Vol. 79, pp. 690-694, January 1982 Neurobiology

Opioids regulate cGMP formation in cloned neuroblastoma cells (cyclic nucleotides/ receptor/desensitization) GERMAINE J. GWYNN AND ERMINIO COSTA* Laboratory of Preclinical , National Institute of Mental Health, Saint Elizabeths Hospital, Washington, D.C. 20032 Communicated by Floyd E. Bloom, October 8, 1981

ABSTRACT caused a rapid dose-related el- cumulation in rat striatal slices with characteristics that fulfill evation of the cGMP content of N4TG1 murine neuroblastoma the criteria for a specific action (6). In contrast, the cells. An excellent correlation was found between the rank order decrements in the cGMP content ofcerebellar tissue observed of of agonists in stimulating cGMP accumulation and in after administration are probably indirect effects of displacing [3H] ([H]ETP) bound to intact cells. The nar- stimulation mediated by the modulation of y- cotic antagonists and failed to increase aminobutyric acid or acetylcholine collateral neuronal loops (7, cGMP content; moreover, in the presence of 5 ,IM naloxone, the 8). A functional role for cGMP in opioid action is further sug- EC50 of ETP increased from w9 nM to >1 ,uM. N4TG1 cells that had been incubated for 20 min with 0.32 ItM ETP and thoroughly gested by the pivotal role that calcium ions play both in regu- washed displayed a marked loss in sensitivity to subsequent ETP lating cGMP formation (for review, see refs. 9, 10) and in me- challenge. This desensitization was characterized by a 40-50% diating the acute and chronic effects ofopioids (for review, see decrease in maximal response and an increase in the apparent Ka refs. 11, 12). Changes in intracellular cGMP content modulate of ETP from 4 to 50 nM. Desensitization was complete after a 7- the activity of cGMP-dependent protein kinases (13). min incubation with 0.32 jIM ETP (t112 1 min) and was only clearly control the phosphorylation of synaptic membrane pro- slowly reversible (t4/2 > 60 min). Naloxone (5 ,AM) and dipren- teins, some of which may be specific substrates for cGMP-de- orphine (0.1 ujM) failed to elicit desensitization, but they blocked pendent kinases (14, 15). ETP-induced desensitization. Dextrophan and (+)-ethylketazo- Clonal cell lines derived from mouse neuroblastoma C1300 cine were <1% as effective as levorphanol and (-)-ethylketazo- exhibit many properties characteristic of neurons (for review, cine, respectively, in both stimulating cGMP accumulation and see refs. 16, 17). The use of such clonal lines as model systems inducing desensitization. When the binding of [3H]ETP (0.2-20 to study the operation ofopioid receptors obviates many of the nM) was examined under identical experimental conditions, cells interpretative complications inherent to studies with intact tis- that were completely desensitized by incubation with ETP (7 min sues. Opioid binding sites are negatively coupled to adenylate with 0.32 ,IM or 20 min with 15 nM) showed no loss ofhigh-affinity cyclase in the neuroblastoma x glioma hybrid line 108CC15 recognition sites. After longer incubation with ETP (0.32 ,AM for (18-20) and in the N4TG1 neuroblastoma clone (21). Studies 20-60 min), the maximal binding of [3H]ETP was reduced purported to show that opioids modify cGMP levels in the hy- 17-41%. The specific short-term desensitization of cGMP accu- brid 108CC15 line (22, 23) have proven to be unreliable (24, 25). mulation is not mediated or accompanied by a decrement in the We report that opioid receptor agonists elicit a rapid dose-re- number of binding sites. lated elevation ofcGMP content in the N4TG1 clone and pres- ent evidence that both this acute response and its subsequent Despite the intensive characterization of high-affinity cellular desensitization exhibit some of the properties ofa specific nar- recognition sites for opioids, our knowledge of the molecular cotic effect. mechanisms by which stimuli are amplified and internalized by these sites remains remarkably meager. In fact, we are still MATERIALS AND METHODS seeking appropriate ways to classify the multiple types of rec- Materials. were from the following sources: [D- ognition, coupling, and amplifying systems associated with the Ala2,D-Leu5], human f3-endorphin, and various types of opioid receptors. Undoubtedly, the endoge- 1-13 (Peninsula Laboratories, San Carlos, CA); naloxone hy- nous population of opioid ligands is heterogeneous and these drochloride (Endo Laboratories, New York); morphine sulfate different molecular forms may well subserve different receptor (Mallinckrodt); and dextrophan tartrates (Hoff- mechanisms. mann-LaRoche); etorphine (ETP) and diprenorphine hydro- Several lines of evidence suggest that the stimuli reaching chlorides (National Institute of Abuse, Rockville, MD); opioid recognition sites are often amplified by changes in the and the compounds (W. Michne, Sterling- formation of cyclic nucleotides and, therefore, by cyclic nu- Winthrop, Rensselaer, NY). 3-Isobutyl-l-methylxanthine (IBMX) cleotide-regulated processes. Numerous studies have shown was from Aldrich. [3H]ETP (specific activity, 32-60 Ci/mmol; that the adenylate cyclase system of the central and peripheral 1 Ci = 3.7 X 1010 becquerels) and 125I-labeled cGMP (2'-O- nervous systems is modified by opioid administration in vivo succinyliodotyrosinemethyl ester) were from Amersham. All and in vitro (for review, see refs. 1-4). The relationship between drug doses were calculated as base content. opioid effects and the guanylate cyclase system has, however, Methods. Stock cultures ofmouse neuroblastoma cells ofthe not been fully explored. doses of morphine cause a N4TG1 clone (a gift from K.-J. Chang, Burroughs Wellcome, dose-related increase in rat striatal cGMP content that is ster- Research Triangle Park, NC) were grown in 250-ml plastic flasks eoselective and blocked by (5). In addition, mor- (Falcon) in Dulbecco's modified Eagle's medium (GIBCO)/ phine and the enkephalin pentapeptides increase cGMP ac- 5% fetal calf serum (GIBCO or M. A. Bioproducts, Bethesda, MD) supplemented with penicillin G at 25 units/ml and strep- The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviations: IBMX, 3-isobutyl-1-methylxanthine; ETP, etorphine. ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. * To whom reprint requests should be addressed. 690 Downloaded by guest on September 26, 2021 Neurobiology: Gwynn and Costa Proc. Natl. Acad. Sci. USA 79 (1982) 691

tomycin at 25 /.g/ml (GIBCO) at 370C in humidified 95% air/ 5% CO2. Cells were subcultured using 1 mM EDTA and grown 80 to confluency (4-6 days) in 60 x 15 mm plastic Petri dishes for use in the cGMP and monolayer culture [3H]ETP binding as- says. In these studies, stationary phase cultures of passages o ~.60 16-29 were used 16 hr after their last feeding. cGMP are Details of methodology for the determinations 40 given in the legend to Fig. 1. For the [3H]ETP binding assays, monolayer cultures were incubated at 370C for various times (1-60 min) in the presence (desensitized cells) or absence (con- trol cells) ofunlabeled ETP (10-320 nM) and washed according 2 20 to the protocol B used for the cGMP determinations. After a 10-min equilibration in 2 ml of medium II (total binding) or medium 11/50 AM levoThanol (nonspecific binding), the cul- 0 1 2 3 4 5 10 15 20 tures were treated with [ H]ETP (0.2-20 nM) for 1 min (unless Time, min otherwise noted). The reaction was terminated by four rapid washes with ice-cold medium II (requiring '10 sec). Subse- FIG. 1. Time course of increase in cGMP content of N4TG1 cells caused by ETP. Monolayer cultures (passage were incubated for 20 quently, 1 ml of 1.0 M NaOH was added to each dish and ali- 19) min at 37°C in medium II and then treated with 0.32 ,M ETP (A) or quots were processed for protein and tritium determinations. 0.9% NaCl (.) (protocol A). Data represent mean ± SEM for 3-5 cul- Specific binding constituted 60-90% of total binding under tures, each assayed in duplicate. This study was repeated twice (pas- these conditions. The SAM 27 program (available from the Na- sages 20 and 21) with similar results. cGMP determinations were car- tional Institutes of Health) was used to calculate the apparent ried out at 37°C. The growth medium was removed and each dish Kj and the maximum number of binding sites. was washed twice with 4 ml of medium I (137 mM NaCl/7.8 mM Na2HPO4/2.7 mM KC1/1.5 mM KH2PO4/0.9 mM CaCl2/0.5 mM RESULTS MgCl2/5.6 mM glucose, pH 7.2). After a 10-min equilibration period, medium I was replaced with 2 ml of medium II (medium 1/0.5 mM Characteristics of the Elevation of cGMP Content Caused IBMX). From this point, protocol B was followed when desensitization by Opiate Receptor Agonists. In N4TG1 cells treated with 0.5 was being measured and protocol A was followed when it was not. In mM IBMX, the addition of 0.32 AM ETP caused a rapid tran- protocol A, cultures were incubated in medium II for 20 min and then sient elevation of intracellular cGMP content (Fig. 1). The treated with the test drug for 1 min. In protocol B, cultures were first in II in presence or cGMP content 70% increase 20 sec, incubated medium the (desensitizing condition) attained ofthe maximal by absence (desensitization control) of various drugs for 20 min, washed peaked at 1 min, and decreased to control values by 5 min. This four times with 4 ml of medium II, and, after a 10-min equilibration rapid decrease is due to a combination offactors, including re- in 2 ml of medium II, treated with the test drug for 1 min. All final drug sidual phosphodiesterase activity, efflux ofcGMP into the me- dilutions were in medium II or 0.9% NaCl and additions were made dium, and desensitization (Table 1). In the absence of IBMX, in 20- to 50-,ul volumes. Forboth protocols, the reaction was terminated the cGMP content ofboth untreated and ETP-treated cultures by aspirating medium II and adding ice-cold 0.4 M perchloric acid. The cGMP content of was ra- was markedly reduced but the difference between the two was neutralized supernatants determined by dioimmunoassay as described (26). Protein was determined as de- in a nM still statistically significant: typical experiment, 10 ETP scribed (27) using bovine serum albumin as standard. Statistical sig- elevated the cGMP content from 13 ± 0.4 to 40 ± 2.6 pmol/ nificance was calculated using Student's t test (two-tailed). mg of protein in the presence of 0.5 mM IBMX and from 4.9 + ± 0.1 to 11 0.7 pmol/mg ofprotein in the absence of IBMX. in displacing [3H]ETP (3 nM) binding to intact N4TG1 cells was Both basal levels of cGMP (5-15 pmol/mg of protein) and the identical. The assays were carried out without peptidase inhib- increase produced by a maximal dose of ETP (0.32 AM; 6- to itors. Naloxone (up to 50 MuM) and diprenorphine (up to 1 uM) 9-fold elevation), but not the EC50 of ETP, varied from exper- failed to elevate intracellular cGMP content. Dextrophan was iment to experiment. All these parameters, however, were <1% as effective as levorphanol and (+)-ethylketazocine was highly consistent within each experiment. The increase in cGMP content caused by acute ETP treat- Table 1. Time course of desensitization of cGMP formation ment was dose-related and antagonized by naloxone (Fig. 2). elicited by ETP in N4TG1 cells A significant increase in cGMP content could usually be de- tected with 0.5 nM ETP and maximal stimulation ofcGMP for- cGMP, mation was elicited by 0.1-0.32 AM ETP. In six independent Addition Time, min ETPchallenge pmol/mg of protein experiments, the EC50 of ETP was consistent, ranging from 8 Buffer - - 7.8 ± 0.2* to 11 nM. Eadie-Hofstee plots of these data show that the K, ETP 1-45 - 7.5 ± 0.1* of ETP was 5.2 nM. After a 5-min treatment with 5 MM nalox- Buffer - + 18.8 ± 1.1 one, the EC50 of ETP shifted from 9.7 nM to 1.1 MM, a >100- ETP 1 + 13.3 ± 0.7 fold increase (Fig. 2). Lineweaver-Burk plots ofthese data con- ETP 3 + 10.8 ± 0.4 firm the competitive nature ofthis antagonism. Very low doses ETP 7 + 8.2 ± 0.3 ofnarcotic antagonists, 40 nM naloxone or 2 nM diprenorphine, ETP 20 + 7.9 ± 0.2 caused significant antagonism ofthe cGMP increase elicited by ETP 45 + 8.3 ± 0.4 10 nM ETP (data not shown). Monolayer cell cultures (passage 26) were incubated in medium II To determine what type of opioid binding site was coupled at 370C for 10 min before addition of 0.32 ,uM ETP or buffer for the to guanylate cyclase in N4TG1 cells, we tested a variety ofopioid indicated time periods. Cultures were washed (protocol B) and then alkaloids and peptides for their ability to increase cGMP content challenged with 10 nM ETP or buffer for 1 min. Data represent mean (Table 2). The rank order of potency of opiate receptor agonists ± SEM for 4-12 cultures. * Average cGMP values of the two control groups (cultures treated in increasing cGMP accumulation was [D-Ala2-D-Leu5]enke- throughout with buffer or challenged with buffer after ETP treat- phalin = ETP > 13-endorphin = dynorphin > (±)ethylketa- ment) were not significantly different at any of the times tested. Data zocine > levorphanol > morphine. The rank order of potency from all times were, therefore, grouped and averaged. Downloaded by guest on September 26, 2021 692 Neurobiology: Gwynn and Costa Proc. Nad Acad. Sci. USA 79 (1982)

80 cultures similarly treated but without ETP in the first incuba- 80 tion (Fig. 3). This desensitization was characterized by a 40-50% decrease in the maximal response elicited by ETP and 60- by a 7- to 12-fold increase in its EC50. Eadie-Hofstee plots of 0 the data from four experiments show that the mean apparent K. of ETP in N4TG1 cells desensitized in this manner was 49.5 40 nM compared with 4.3 nM in control cultures. The washing procedure removed >99% ofthis first dose of ETP (1.6 nM to- 20 tal, 0.5 nM specifically bound to cell layer remained). Desen- = 20t sitization failed to affect the time-course of the increase in cGMP content; peak effect still occurred at 1 min. Desensiti- zation could be completely blocked by prior treatment with 5 10-10 10~- 10-8 10-7 10-6 i0-5 ,uM naloxone (Fig. 3). The extent ofdesensitization was dependent on the concen- ETP, M tration and nature of the opioid present during the first incu- FIG. 2. Dose-response curves of cGMP accumulation elicited by bation (Fig. 4). Treatment with 50 pM ETP failed to desensitize ETP in the presence and absence of naloxone. Monolayer N4TG1 cul- to a challenge with 10 nM ETP, while 0.32 ,uM ETP caused tures (passage 22) were incubated at 370C in medium II for 15 min and complete desensitization. The ED50 for ETP for inducing de- then for 5 min in the presence (A) or absence (A) of naloxone (5 ,uM) sensitization was -5 nM. Doses of 50 ,iM levorphanol and 10 before treatment with various doses of ETP for 1 min. Data represent ,uM (-)-ethylketazocine were roughly equipotent with 0.32 mean ± SEM for four or five cultures. Mean control cGMP levels (i.e., ,uM ETP in causing almost complete desensitization. In con- cGMP content of cultures not stimulated with ETP) in untreated and naloxone-treated cultures were not significantly different (8.7 and 9.0 trast, the less active stereoisomer dextrophan (50 ,uM) did not pmol/mg of protein, respectively). This experiment was repeated six cause desensitization and (+)-ethylketazocine (10 ,uM) caused times with similar results. only 17% desensitization. The presence of naloxone together with ETP during the first incubation blocked the development ofdesensitization (Fig. 3). However, prior incubation with nal- <0.3% as effective as (-)-ethylketazocine in stimulating cGMP oxone (5 ,uM) or diprenorphine (0.1 ,uM) alone surprisingly formation. These isomers displaced [3H]ETP binding to intact caused significant (P < 0.05) potentiation ofthe cGMP response N4TG1 cells with similar potency ratios. to 10 nM ETP (Fig. 4). Effects of Prior Short-Term Opioid Treatment on the In- Desensitization ofcGMP accumulation was a function ofthe crease in cGMP Content Elicited by Opioid Receptor Stimu- length ofprior incubation with ETP (0.32 ,tM). Marked refrac- lation. N4TG1 cells that had been incubated for 20 min with toriness to a subsequent ETP challenge (10 nM) developed after 0.32 ,uM ETP and thoroughly washed displayed a marked loss a 1-min incubation and maximal desensitization was evident on in sensitivity to subsequent ETP challenge when compared with treatment -7 min (Table 1). Responsiveness to ETP challenge returned with a tl/2> 60 min (Table 3). Table 2. Potency of opioid agonists in stimulating cGMP Comparison by Scatchard Analysis of the Opioid Recogni- formation and in competing with the binding of [3H]ETP in intact tion Sites Labeled by [3H]ETP in Intact Control and Desen- N4TG1 cells sitized N4TG1 Cells. Using conditions identical to those used Stimulation* Displacementt in the cGMP assays, we compared the binding of a wide range Opioid (EC50), nM (IC50), nM of concentrations of [3H]ETP to monolayer cell cultures that [D-Ala2-_-Leu5]enkephalin 10 4.7 ETP 11 3.2 900 ,l3Endorphin (human) 90 12

Dynorphin (1-13) 76 15 _-4 (-)-Ethylketazocine 165 17 -b 700 (-Ethylketazocine 380 28 0 Levorphanol 1,800 75 0 Morphine >100,000 >600 e, 500 (+)-Ethylketazocine >50,000 >5,000 Dextrophan >200,000 > 10,000 Q 300 * Monolayer cultures (passage 18 or 27) were incubated at 370C for 20 min in medium II and then stimulated for 1 min with opioid. Three to five cultures were used at each of the three to six concentrations 100 -1 of opioid tested. 10-10 10-9 10-8 10-7 10-6 10-5 t Cultures on 150 x 25 mm plastic dishes (passages 16 or 27) were washed twice with medium I and gently scraped from the dish with ETP, M a Teflon spatula. Binding assays were conducted in triplicate in 12 x 75 mm plastic tubes (250-500 ,g of protein per tube) in (total vol, FIG. 3. Desensitization of the ETP-caused elevation of cGMP con- 0.5 ml) medium I1/0.4% bovine serum albumin. Specific binding is tent and antagonism of desensitization by naloxone. Monolayer cell defined as the difference in binding in the absence and presence of cultures (passage 25) were incubated at 3700 for 20 min in medium I 10 AM unlabeled ETP. After 25 min at 370C, binding was terminated (9), medium 11/0.32 ,uM ETP (A), or medium II/0.32 ,uM ETP/5 ,uM by adding 4 ml of ice-cold medium II to each tube and filtering the naloxone (A). They were then washed (protocol B) and treated with contents through a Whatman GF/B filter (2.4 cm) at reduced pres- various doses of ETP for 1 min. Data represent mean + SEM for five sure. Each filter was rapidly washed three times with 4 ml of ice-cold cultures. Mean control cGMP levels (i.e., cGMP content of cultures not medium II and assayed at least 16 hr later using liquid scintillation stimulated with ETP) in untreated cultures and cultures previously spectrophotometry at 40-45% efficiency. The IC50 of the specific treated with ETP or ETP/naloxone were not significantly different binding of [3H]ETP (3 nM) was estimated from competition curves (14.6, 13.8, and 15.1 pmol/mg of protein, respectively). This experi- using four to seven different concentrations of test opioid. ment was repeated four times with similar results. Downloaded by guest on September 26, 2021 Neurobiology: Gwynn and Costa Proc. Natl. Acad. Sci. USA 79 (1982) 693

-I, binding (4 nM) when tested immediately after washing but a 400 1l significantly greater decrease (34%) when tested 40 min later. 1z- Because ETP elicited a maximal accumulation of cGMP vTI within 1 min (Fig. 1), we examined the time course of[3H]ETP o300 binding (2 nM and 10 nM) in monolayer cultures ofcontrol and -a desensitized cells (0.32 ETP for 20 min). Binding was ex- 0 kkM tremely rapid < 20 sec) and, in both conditions, was similar 200 (tl/2 k=- after a 1-min versus a 25-min incubation with label. In agree- ment with the results of the Scatchard analyses, at 2 nM and 10 nM [3H]ETP, the amount of ligand specifically bound to Y. 100 r desensitized cells was 82% and 103%, respectively, of that ob- 0 X ai (a served in control cultures. to eq cq 0 .C) 0 to 10 m'lID 10 1- 1 2 3 4 5 6 7 8 9 10 11 12 13 DISCUSSION Drug used Since the elevation ofcGMP content elicited by opiate receptor CQ agonists in N4TG1 cells occurs in the presence of 0.5 mM FIG. 4. Effects of prior incubation with various opioidagonists and IBMX, which inhibits the cGMP-dependent phosphodiesterase antagonists on EPT-induced increase in cGMP accumulation. Mono- activity present in this (unpublished observations) and related layer cultures of N4TG1 cells (passages 17, 18, and 20) were incubated clones of neuroblastoma cells (28, 29) by >70%, we infer that for 20 min at 370C in medium II in the presence or absence of drug. in reflects stimulation Cultures were washed (protocol B) and then treated for 1 min with 10 this increase intracellular cGMP content nM ETP. Data represent mean ± SEM for four or five cultures. Prior of guanylate cyclase activity rather than inhibition of cGMP treatment did not significantly affect basal cGMP levels. These ex- degradation. Both the acute stimulation and the subsequent periments were repeated once with similar results. Drugs used: 1, 5, desensitization of cGMP formation by opioids fulfill the com- and 8, none; 2, 0.5 nM ETP; 3, 5 nM ETP; 4 and 9, 0.32 MxM ETP; 6, 5 monly acknowledged criteria for a specific narcotic action. They ,uM naloxone; 7, 0.1 uM diprenorphine; 10, 50 MLM levorphanol; 11, 50 had the same > > lev- MM dextrophan; 12 and 13, 10 AM (-) and (+)-ethylketazocine, agonist selectivity (ETP ethylketazocine respectively. orphanol) and displayed marked stereoselectivity. Naloxone and diprenorphine neither stimulated nor desensitized guanylate were either untreated or previously treated with ETP (10 cyclase activity but blocked the actions ofETP. The EC50ofETP nM-0.32 ,AM) for various periods of time (1-60 min). In un- for inducing desensitization (-5 nM) was similar to the EC50 treated cells, Scatchard plots were nonlinear, consistent with of ETP for stimulating cGMP accumulation (8-11 nM). Inter- two populations of sites, with Kd and maximal binding param- estingly, however, the time courses for these two processes eters of 1.1 ± 0.4 nM (95 ± 12 fmol/mg of protein) and 10.2 were different: 0.32 p.M ETP elicited peak elevation of cGMP + 2.3 nM (260 ± 38 fmol/mg ofprotein). Hill plots ofthese data content after 1 min (Fig. 1) but required -7 min to induce com- showed two linear portions with coefficients of 0.9 ± 0.1 and plete desensitization (Table 1). Taken together, these data in- 1.5 ± 0.2. ETP treatment that induced significant (0.32 AuM for dicate that the desensitization process is initiated by agonist 1 min; Table 1) or maximal desensitization (0.32 ,uM for 7 min occupancy ofthe opioid recognition sites present on these cells. or 15 mM for 20 min) of the cGMP response failed to signifi- This proposition is supported by the fact that the apparent K3 cantly modify the binding properties of [3H]ETP. However, values for [3H]ETP binding to intact N4TG1 cells (I.3 1 and prior incubation for 20-60 min with 0.32 ,uM ETP caused a 10 nM) correlate well with the apparent activation constant of progressive decrease (17-41%) in the number of high-affinity ETP for cGMP accumulation (K.- 4 to 5 nM). Furthermore, binding sites. Interestingly, this decrease in the number of in a series of alkaloid and peptide agonists, the rank order of [3H]ETP recognition sites, once initiated, was not dependent potency in displacing [3H]ETP binding (3 nM) was similar to on the-continued presence of agonist: compared with untreated that for stimulating cGMP accumulation. cells, cultures previously treated with ETP (0.32 ,uM for 20 min) Although specific antagonists of the purported subtypes of and washed free of agonist showed a 17% decrease in [3H]ETP opioid receptors are not available to allow a clear characteriza- tion of the type of receptor that is operative, our results (Table Table 3. Time course for reversal of ETP-induced desensitization 2) indicate that in N4TG1 cells a delta type of recognition site of cGMP formation in N4TG1 cells is positively coupled to guanylate cyclase. Scatchard plots of [3H]ETP binding (after a 1-min or a 25-min incubation at 37°C) Incubation to intact N4TG1 cells were curvilinear. These observations are ETP after washing, ETP cGMP, pmol/ consistent with, but do not distinguish among, at least four dif- treatment min challenge mg of protein ferent models: receptor heterogeneity, negative cooperativity, - 0-60 - 7.0 ± 0.2* dissociable receptors, and a two-step interaction (30). A similar + 0-60 - .6.8 ± 0.3* type of delta recognition site for opioids appears to mediate in- - 0-60 + 22.9 ± 1.7* hibition of adenylate cyclase in both the 108CC15 (31) and + 0 + 8.8±0.5 N4TG1 clonal lines (21). In both cell lines, opioids also regulate + 15 + 9.1±0.3 the biosynthesis of various glycoproteins and gangliosides (32), + 30 + 10.3 ± 0.4 which are thought to be components of receptors and ionic + 60 + 13.0 ± 0.4 channels in some cells (33, 34). Monolayer cell cultures (passage 26) were incubated in the presence There are significant qualitative differences in the hormonal or absence of 0.32 MM ETP and then washed according to protocol B. regulation of cyclic nucleotides in the 108CC15 and N4TG3 At the indicated times after washing, cultures were challenged with lines (35). N4TG3 and N4TG1 are 6-thioguanine-resistant mu- buffer or10 nM ETP for 1 min. Data represent mean ± SEM for 4-12 tants from the same neuroblastoma N4. As the cultures. derived clone, * In each of these conditions, average cGMP values obtained at differ- hybrid line (36) was derived by fusion with gliomacells (N18TG2 ent times after washing (0-60 min) were not significantly different mouse neuroblastoma x rat glioma C6-BU-1), N4TG1 cells may and, therefore, the mean of the grouped data is presented. provide a more physiologically relevant neuronal model. To Downloaded by guest on September 26, 2021 694 Neurobiology: Gwynn and Costa Proc. Natl. Acad. Sci. USA 79 (1982) date, we have not extended our studies to the 108CC15 hybrid 9. Rasmussen, H. & Goodman, D. B. P. (1977) Physiol Rev. 57, line. However, preliminary experiments show that a large dose 422-509. of ETP (0.32 ,uM) caused a significant elevation of the intra- 10. Goldberg, N. D. & Haddox, M. K. (1977) Annu. Rev. Biochem. 46, 823-896. cellular cGMP content of NlE115 neuroblastoma and PC12 11. Ross, D. H. & Cardenas, H. L. (1979) Adv. Biochem. Psycho- pheochromocytoma cells but was ineffective in the NB2a neu- pharmacot 20, 301-338. roblastoma and C6 glioma lines, which lack a significant pop- 12. Chapman, D. B. & Way, E. L. (1980) Annu. Rev. Pharmacol Tox- ulation of opioid binding sites. icol 20, 553-579. To gain insight into the possible mechanisms underlying 13. Greengard, P. (1978) Science 199, 146-152. opioid desensitization, in other studies we have examined 14. Ehrlich, Y. H., Davis, L. G., Keen, P. & Brunngraber, E. B. (1980) in Endogenous and Exogenous Opiate Agonists andAntag- whether crossdesensitization would develop between ETP and onists, ed. Way, E. L. (Pergamon, New York), pp. 229-232. nonopioid agents that also elevate the cGMP content ofN4TG1 15. Clouet, D. H. & O'Callaghan, J. P. (1979) Adv. Biochem. Psy- cells (unpublished). No crossdesensitization was observed be- chopharmacot 20, 281-300. tween ETP and histamine, carbachol, sodium azide, man- 16. Breakefield, X. 0. (1976) Life Sc. 18, 267-278. ganese, or A23187. These data suggest that opioid desensiti- 17. Hamprecht, B. (1977) Int. Rev. Cytol. 49, 99-170. zation is not due to 18. Klee, W. A. & Nirenberg, M. W. (1974) Proc. Natl. Atad. Sci. (i) alterations in the specific activity ofeither USA 71, 3474-3477. guanylate cyclase or cGMP-phosphodiesterase or (ii) inactiva- 19. Sharma, S. K., Klee, W. A., & Nirenberg, M. (1975) Proc. Nati. tion of calcium entry (37). Acad. Sci; USA 72, 590-594. Maximal desensitization of the cGMP response can be ob- 20. Traber, J., Fischer, K., Latzin, S. & Hamprecht, B. (1975) Na- tained with ETP treatments that fail to alter the Kd or maximal ture (London) 255, 558-559. binding value of the opioid receptor for [3H]ETP. Treatments 21. Miller, R. J., Dawson, G. & MacLawhon, R. (1980) in Endoge- with 0.32 ,uM ETP lasting longer than 20 nous and Exogenous Opiate Agonists and Antagonists, ed. Way, min, however, re- E. L. (Pergamon, New York), pp. 267-270. sulted in significant loss ofagonist binding sites. In contrast, no 22. Gullis, R. J., Traber, J. & Hamprecht, B. (1975) Nature (London) decrement or internalization of ['5I]-labeled [D-Ala2-D-Leu5]- 256, 57-59. enkephalin binding sites of N4TG1 cells was reported to occur 23. Brandt, M., Gullis, R. J., Fischer, K., Buchen, C., Hamprecht, after 24 hr of morphine treatment (38, 39). Although the kd of B., Moroder, L. & Wunsch, E. (1976) Nature (London) 262, ETP did not change appreciably during desensitization, the Ka 311-313. 24. Gullis, R. J. (1977) Nature (London) 265, 764. for elevating cGMP content increased >10-fold. This shift is to 25. Hamprecht, B. (1977) Nature (London) 265, 764. be expected in all systems in which the coupling between site 26. Harper, J. F. & Brooker, G. (1975) J. Cyclic Nucleotide Res. 1, occupancy and enzyme stimulation is not linear (40) and indi- 207-218. cates that a change in the coupling function occurred during 27. 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