Prog. Neuro-Psychopharmocol. 6 Biol. Psychiot. 1984. Vol. 8. pp. 719-723 0278-5846/M 50.00 + .50 Printed in Great Britain. All rights reserved Copyright 0 1984 Pergamon Press Ltd.

OPIATE PEPTIDE RECEPTORS ON INTACT NIE-115 NEUROBLASTOMA: RADIOLIGAND BINDING PROPERTIES, INTRACELLULAR RESPONSE, AND

EFFECTS OF INCREASING MEMBRANE CHOLESTEROL

BARBAR?+ G. FAO' AND MARY G. MURPHY2

1 Biology Department, Mt. St. Vincent University and

2 Departinent or Pharmacology, Daihousie

University, Halifax, Nova Scotia, Canada

(Final form, June 1984)

Abstract

Rao, BaKbaKa G. and Mary G. Murphy: Opiate peptide receptors on intact NlE-115 nauroblas- tcma: radioligand binding properties, intracellular response, and effects of increasing membrane cholesterol. Prog. Neuro-psychopharmacol. & Bio. PsychiatK. 1984, 8 (4-6): 719-723. -

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3. 4.

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Binding of [3H]methionine-enkephalin to intact NlE-115 neuroblastoma cells (competing ligand: naloxone) revealed a homogenous population of receptors with a density (ammo) of 79.U + 6.5 fmol/nq protein (me?" SFM, N=3) and an apparent Kd of 5.33,~ 1.63 mM. The order of displacement of [ H]met-enkephalin was Lmet-/leu-enkephalrn > naloxone > ,norphine, suggesting that it is of the delta receptor class. Specific binding was heat-labile, stereospecific and sensitive to Na+. Adding met-enkephalin to intact neuroblastoma caused reductions of both basal and prostaglandin E -stimulated levels of cyclic AMP (41.4 + 4.8% (N=6) and 45.1 + 2.4% (N=3) of contra 4 levei$, respectively). Maximum inhibition (naloxone-reversiE1e) was observed as low as 1W M met-enkephalin. Preliminary results syggest that cells grown in cholesterol-supplemented medium show reduced binding of [ HImet-enkephalin.

Keywords: cholesterol, cyclic AMP, met-enkephalin, NlE-115 neuroblastona, opiate peptides.

Abbreviations: Dulbecco's Modifiec Eagle's Medium (DMXM), N-2-hydroxyethylpiperazine-N'-2- ethanesulfonic acid (HEWS), phosphaticiylcholine (PC), prostaglandin E 1 (xE1).

Introduction

A hcxaogenous class of delta-type fully functional opiate receptors has been shown in cultured neuroblastcma (Gilbert et al., 1982; Law et al., 1982a and 198219) and neuroblas- tcnna x glioma hy$id cells (Gi&beKt et al., 1982 and Sharma et al., 1975) by use of radio- ligands such as [ Hlmethionine -enkephalin. Occupation of the receptors by active opioid peptides results in a dose-dependent inhibition of both basal and prostaglandin E &GE )- stimulated adenylate cyclase. Direct correlation between ligand-receptor interac ions &' and intracellular responses has not teen possible in most studies of opioid receptors on cul- tured cells, since the two are rarely examined under similar experimental conditions. Although changes in accumulation of cyclic AMP are frequently measured in intact cells, binding assays are often done with .msmbrane fragments. To date, the only investigation of properties of binding of radiolabelled opioid peptides to NlE-115 neuroblastolna cells used cell homogenates in TKiS buffer at O" for 2 hr (Gilbert et al., 1982).

We now report the properties of binding of [3H]met-enkephalin under conditions that do not compromise cell viability. Under these conditions, met-enkephalin elicits a dose-dependent inhibition in FGE -stimulated adenylate cyclase activity. evidence that bid'

FUKtheKnOKe, we hdVt? preliminary

ing can be altered in cells supplemented with cholesterol.

719

720 B. G. Rao and M. G. Murphy

Methods

Materials: [3H]meti~ionine5-enkephalin was obtained from New England Nuclear. Displacers were obtained as follows: morphine sulfate, BDH; levorphanol and dextrorphan, Hoffman- LaRoche Ltd.; naloxone, met-enkephalin and leu-enkephalin, Sigma Chemical CO. Ro20-1724 (4- (3-butoxy-4-rrurthoxybenzyl)imidazolidin-2-one) MS from Hoffman-LaRoche. Other chemicals were purchased frcan Sigma Chemical Co., or Canlab or Fisher Scientific Ltd.

Cell culture: -- Mouse neuroblastoma, clone NlE-115, mre grown according to Murphy (1984). Cells were harvested 3-5 days after subculture by gentle flushing, centrifuged, and washed twice with 5 mM HEPES-HCl buffer, pH 7.6, containing 0.5 mM MgC12, sucrose.

0.5 mM CaC12 and 0.32 M Viability was then assessed by trypan blue exclusion ( >80% viable). Protein was

determined using bovine serum albumin as standard (Lowry et al., 1951). When membrane cholesterol content was manipulated, solutions of cholesterol/phosphatidylcholine (Shattil et al., 1975) in ratios of 2 (high cholesterol), 1 (medium) or 0 (PC only) wre added to growth medium 24 h before harvest.

assays: Binding Cell suslpnsions (0.4-0.8 irg protein) ware incubated in a final vole of 1 ml HEPES-HCl containing [ HImet-enkephalin (5-40 WI), 10 i.lK puromycin, + displacing drug. Naloxone (10 FiM) w%s rogtinely used. Incubation was initiated by addition of cells, con- tinued for 20 min at 30 C in a shaking water bath and terminated by filtration through Whatman CF/B glass fiber filters, with three rinses of ice-cold Tris-Xl buffer (50 mM, pH 7.4). Filters were dried and radioactivity cgunted in a Mark III liquid scintillation counter. Specific binding is the amount of [ HImet-enkephalin bound to the cells in the absence of displacer minus the amount bound in the presence of displacer.

Cyclic AMP measurement: Adenylate cyclase activity was examined by measuring accumulation of CAMP according to the methods cited in Murphy (1984).

Results

The time course for specific binding of [3H]rret-enkephalin in the presence of 10 FM naloxone varied with temperature (Fig. 1). Initial rates were sloer at lowar temperatures but higher maximum levels of specific binding were observed, a phenomenon which has been reported @hang et al., 1978) and may reflect faster degradation of the peptide at 37'C.

EFFECT OF ,NCUBATION TEMPERATURE ON TIME

COURSE 0 F BINDING OF %-MET - ENKEPHALlN

TIME (min #

Fig. 1. Time (min)oand tenl%rature ( n = 25’~; . = 30 c; A =37 C) d5pendence of specific binding of 5r-u~ [ )l]met-enkephalin (flnole/mg protein) to intact NlE-115 rwroblastoma.

SATURABLE BINDING OF %i-MET - ENKEPHAUN

~0 ,NTACT N,E - 115 NEUROBLASTOMA

%-MET - ENKEPHALIN (nM)

Fig. 2. Amount of [3H]rret-enkephalin specifically bound to intact NlE-115 at NlE-115 at various concentrations of ]3H]~t-enkephalin (nM). Inset: Scatchard analysis.

Opiate receptor function in NIE-115 neuroblastoma 721

At 30'C for 29 min, specific binding was 23.4 + 2.5 fmol/mg protein at an average concentra- tion of 5nM [ Hlmet-enkephalin or approximately 40% of total binding. Specific binding incgeased linearly with protein to at least 1 m/ml and was abolished by heating cells at 100 C for 5 min before assay. Data from several experiments yielded a 3 = 79.0 + 6.51 fmol/mg protein (mean + SEM, N=3) and an apparent K ative saturation curve-is shown in Fig. 2.

value of 5.33 kl.6y&. A rebresent- 2ince t ere are 1.2 x 10 cells/q protein in d

this cell line, this is equivalent to 4 x 10 receptor sites per cell. This density com- pares to that observed in 1982).

other lines (Chang et al., 1978; Law et al., 1979; Gilbert et al.,

The relative potencies reported to be enkephalin

of opiates in inhibiting binding to delta-type opioid receptors is > naloxone > morphine (Chang et al., 1982). The results shown in

Figure 3 confirm that the receptors are delta type. Stereospcificity is indicated by the greater potency (1000X) of levorphanol as displacer than its stereoisomer dextrorphan (not shown).

Binding was markedly reduced in the presence of sodium; but s&$um is required for H;E - dependent stimulation of CAMP accumulation. In the absence of Na , levels of CAMP (t F& ) were essentially,the same (111.3%) as those in control cells. (1-100 m"r) of Na (+ PGEl) resulted in a dose-

Increasing the concentrate .J n

tide concentration (not shown). Addition of 10 _$penden_t7increase (up to 8-fold) in nucleo-

to 10 dependent accumulation of CAMP with an ICsO of 7.5 x 10

_6M met-enkephalin inhibited FGEl- M (Fig. 4).

DISPLACEMENT OF 3H-MET - ENKEPHALIN

FROM INTACT NEUROSLASTOMA

- LOG CONCENTRATION (M)

MET-ENKEPHALIN INHlSlTlON OF

PGEq-STIMULATED CAMP FORMATION

9 8 I 6 I . MET-ENKEPHELIN (-LOG CONCENTRATION)

Fig. 3. Displacenent of [3~]-met- enkephalin (5-10nM) from intact NlE-115 neuroblastoma by met-enkephalin ( l ) morphine sulfate (A) or naloxone (m).

Fig. 4. Effect of increasing concentrations of met-enkephalin on FC;I;l-stimulated adenylate cyclase in N~E-115 wuroblastoma.

In cholesterol manipulation experiments, viability of cells fed high or medium cholesterol was comparable to that of untreated cells (approx. 80%). Cells exLased to PC alone were defonned and fragmented and viability did not exceed 20%. Preliminajy results sugcjest that cells supplemented with medium cholesterol show reduced binding of [ Ir]mct-enkephalin, but that displacement is unimpai.red. In cells fed high cholesterol, both binding and displace- ment decreased.

722 B. G. Rae and M. G. Murphy

Discussion

Tne bindin of centrally-acting dKUgS and neUKOtranSmitteKS iS iI%t frequently charactcrizeti usin fragmented meni~rane preparations. Discrepancies between data obtained with nomo- yenates and those obtained with intact cells strongly suggest that. membrane integrity has important eEEects on the properties of receptors, particularly opiate Kect?ptOKS. Investigators have shown that cellular disruption affects displacement potency (Blune et al., 1977) and sensitivity to metal ions (Sadee et al., 19ti2), guanine nucleotides (Rosenbaum and Saoee, lYd3) and proteolytic ano lipolytic enzymes (Chary et al.,lY78). Law et al. (1982) report evidence that cellular integrity is required for maximm inhibition by oyioici Teptides of PGE -stimulated adenylate cyclase activity.

,B We have taken care in these

stuuies to use conditi ns permitting maintenance of cell membrane activity. We have roeas- ureci opioid binding ano inhibition of PGE -stimulat& adenylate cyclase under conditions as similar as Lpossible for both assays. We $ eel that this is essential in order to correlate ligano-receptor interactions and 3 'ntracellular responses witn receptor occupation. Under these conditions, wz found that [ ~]met-enkephalin binding is time- and temperature- dependent, linearly related to protein and cell number, high-affinity, saturable, and stereoqpecific. The order of potency of competing liyands suggests that the NlE-115 opiate receptor is of the delta subtyF,+in agreement with previous reports. Reduction in agonist bindiny to opiate receptors by Na has teen observed in rat brain preparations (Simon et+ al., 1975) and in cultured hGl08-15, but not in N18TG2 0211s (Law et al., 1982). The Na rtquirenent for FGEl stimulation of adenylate cyclase activity is well-documented (Licntshtein et al., 1979). Establishing the properties of [ H]met-enkephalin binding to NlC-115 reuroblastoma was essential since there are important differences in binding and opiate-nrciiateo inhibition of cyclase activity in different cell lines (Law et al., 1982). The results of this study show that this cell line is a good model for studying opiate receptor function under conditions where meaningful correlations can be made between ligand binding data and the intracellular biochemical changes caused by receptor occupation. Current work focuses on the effects of manipulation of the lipid content of the cell membrane on these properties.

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CHANG, K.-J., MILLER, R-J., AND CUATRIXASAS, P. (1978) Interaction of enkephalin with opiate receptors in intact cultured cells. Mol. Pnarmacol. 14: 961-970.

LAW, P.Y., IIEKZ, A. and WH, H.H. (1979) Demonstration and chzacterization of a stereospecific opiate receptor in the neuroblastcma ~18TG2 cells. J. Neurochem. 33: 1177- - 11&7.

LAW, P.Y., HOPl, D.S., and LOH, H.H. (1982) Loss of opiate receptor activity in neuroblastoma x ylioma NS108-15 hybrid cells after chronic opiate treatment: a multiple- step process. Mol. Phannacol. 22: l-4.

WI, P.Y., KOBHLER, J.E. and LOH,~.H. (1982) Comparison of opiate inhibition of adenylate cyclase activity in neuroblastoma Nl8lG2 and neuroblastoma x glioma NG108-15 hybrid cell lines. Mol. Phannacol. 21: 483-491.

LAW, P.Y., NICKSIC, T.D. O?%XlRKE, M.A., KOEHLER, J.E., HERZ, A. and IOH, H.H. (1982) Potentiation of opiate action in neuroblastoma N18TG2 cells by lipid incorporation. Mol. Pharmacol. 21: 492-502.

LICHTSHTEIN, x, BCONE, G., and BLUME, A.J. (1979) A physiological requirement of Na+ for the regulation of c&YP levels in intact NG108-15 cells. Life Sci. 25: 985-991.

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MURPHY, M. (1984) Increasing membrane polyuzturated fatty acid content augments cyclic AMP formation and prostaglandin production in NlE-115 neuroblastcma. J. Prog. Neuro- psychophannacol. & Bio. Psychiatr. 8: in press.

ROSENBAUM, J.S. and SADEE, W. (1983) Opiate receptor binding affected by guanine nucleotide: partial loss of sensitivity to guanosine-5'-(8,y-imido) triphosphate evident in vitro. J. Biol. Chem. 258: 1419-1422.

SADEE, W., PFEIFFER, A., and=RZ, A. (1982) Opiate receptors: multiple effects of metal ions. J. Neurochem. 39: 659-667. -

Opiate receptor function in NIE-115 neuroblastoma 723

SHARMA, S.K., KLEE, W.A. and NIRENBERG, M. (1975) Dual regulation of adenylate CyClaSe accounts for narcotic dependence and tolerance. Proc. Natl. Acad. Sci.U.S.A. 72: 3092- 3096.

SM'ITIL, S.J., ANAYA-GALINDO, J.B., COLMAN, R.W. and COOPER, R.A. (1975) Platelet hypersensitivity induced by cholesterol incorporation. J. Clin. Invest. 55: 636-643.

SIMON, E.J., HILLER, J.M., GROTH, J. and EDELMAN, I. (1975) Further properces of stereosPecific opiate binding sites in rat brain: on the nature of the scdium effect. J. Phannacol. and Exper. Ther. 192: 531-537. -

Inquiries and reprint requests should be addressed to:

Dr. Mary G. Murphy Department of Pharmacology Dalhousie University Halifax, Nova Scotia Canada B3H 4H7