Brain Research, 493 (1989) 23-32 23 Elsevier

BRE 14656

Guanine nucleotide regulation of [125I]fl-endorphin binding to NG108-15 and SK-N-SH cell membranes: specific cation

requirements

Dana E. Selley and Jean M. Bidlack

Department of Pharmacology, The University of Rochester, School of Medicine and Dentistry, Rochester, NY 14642 (U.S.A.)

(Accepted 27 December 1988)

Key words: Guanosine triphosphate; fl-Endorphin; Neuroblastoma cell; Opioid receptor, p-; Opioid receptor, 6-; Opioid peptide

Regulation of [~:5I]flh-endorphin binding by guanine nucleotides was investigated in membrane preparations from two opioid receptor-containing cell lines: NG108-15, which contains only b opioid receptors, and SK-N-SH, which contains predominantly # opioid receptors. In contrast to the binding of the b-selective agonist [3H][D-penicillamine2,D-penicillamineS]enkephalin to NG108-15 cell membranes, and of the p-selective agonist [3H][D-Ala2,MePhe4,Gly-olS]enkephalin to SK-N-SH cell membranes, [125I]fl h- endorphin binding to NG108-15 and SK-N-SH cell membranes was not altered by guanosine triphosphate (GTP) or guanylyl-5"-imidodiphosphate (Gpp(NH)p) in the absence of cations. However, in the presence of NaCI, [125I]flh-endorphin binding to both cell lines was inhibited by GTP and Gpp(NH)p in a concentration-dependent manner. In SK-N-SH cell membranes, the ability of sodium to promote regulation of [125I]flh-endorphin binding by GTP was mimicked by the monovalent cations lithium and potassium, but not by the divalent cations magnesium, calcium, or mangenese. In NG108-15 cell membranes, only sodium was effective in promoting inhibition of [125I]flh-endorphin binding by GTP. The effect of GTP or Gpp(NH)p in the presence of sodium was also observed with guanosine diphosphate, but not guanosine monophosphate or any of the non-guanine nucleotides tested. These results indicate that the presence of monovalent cations is required for regulation of [~25I]flh-endorphin binding by guanine nucleotides, and that the specificity of this cation requirement differs between the p and b receptor-containing cell lines.

INTRODUCTION

Current evidence suggests the involvement of guanosine triphosphate (GTP)-binding regulatory proteins, termed N or G proteins, in the coupling of p and 6 opioid receptors to a variety of second

messenger/effector systems, including inhibition of adenylate cyclase activity ~'23"32, stimulation of guanyl-

ate cyclase activity 24, inhibition of calcium conduc- tance ~6, and stimulation of potassium conductance 28.

The inhibitory effect of guanine nucleotides upon opioid agonist binding 4"5'9-12'3°'35"36 is thought to be

a result of this interaction of opioid receptors with the Ni protein ~s'32'33. Although the endogenous

opioid peptide fl-endorphin has been shown to bind to B and 6 opioid receptors 22 and to inhibit adenylate cyclase activity 3A9'25'26, we have shown that human

[125I-Tyr27]fl-endorphin ([125I]flh-endorphin) binding

to rat brain membranes was not inhibited by guanine nucleotides in the absence of cations 31. However , in

the presence of monovalent cations at concentra- tions sufficient to inhibit [12sI]flh-endorphin binding by themselves, [125I]flh-endorphin binding was sup-

pressed by GTP and guanylyl-5"-imidodiphosphate (Gpp(NH)p) in a concentrat ion-depenent manner. The order of potency of the monovalent cations in

promoting inhibition of [125I]flh-endorphin binding by GTP was: sodium > lithium > potassium. The

divalent cations, magnesium, calcium, and mange- nese were not effective in promoting inhibition of [125I]flh-endorphin binding by GTP, even though the divalent cations alone were potent inhibitors of binding.

In this report, we investigated the regulation by

Correspondence: J.M. Bidlack, Department of Pharmacology, University of Rochester, School of Medicine and Dentistry, Rochester, NY 14642, U.S.A.

0006-8993/89/$03.50 ~ 1989 Elsevier Science Publishers B.V. (Biomedical Division)

24

guanine nucleotides of [125I]flh-endorphin binding to membranes prepared from two opioid receptor- containing cell lines: the neuroblastoma × glioma NG108-15 hybrid cell line, which contains only b-type opioid receptors 8 and the SK-N-SH human neuroblastoma cell line 34. This cell line has been reported to contain predominantly p-type opioid receptors, along with a small number of b recep- tors 17"2°'34. The effect of guanine nucleotides on the

binding of the b-selective opioid agonist, [3H][D-pen- icillamine2,D-penicillamineS] enkephalin (DPDP-E) 2' 14,22.27 to NG108-15 cell membranes, and of the

p-selective opioid agonist, [3H][D-Ala2,MePhe4,Gly - olS]enkephalin (DAGO) 15, to SK-N-SH cell mem- branes was also investigated for comparison with

[125I]flh-endorphin.

MATERIALS AND METHODS

Cell culture Cells were cultured at 37 °C in a humidified

atmosphere of 5% CO 2 and 95% air in EX-CELL 300 medium containing 5% fetal bovine serum, 100 units/ml penicillin, and 100 pg/ml streptomycin. For the NG108-15 cells, 100 pM hypoxanthine, 0.4 pM aminopterin, and 16 pM thymidine were also in- cluded in the medium. NG108-15 cells were har- vested by agitation, while the SK-N-SH cells were harvested by replacement of the medium with ice-cold Ca2+/Mge+-free phosphate-buffered saline containing 0.4% EDTA (PBS-EDTA) and detach- ment of the cells by agitation after incubation in the PBS-EDTA at 25 °C for 5 min.

Cell membrane preparation Cells were harvested as described above and

centrifuged at 200 g for 10 min. The pellet was resuspended in 50 vols of 50 mM Tris-HCl, pH 7.5, and homogenized with a Brinkmann polytron. The homogenate was incubated at 37 °C for 30 min to remove endogenous opioid peptides, and subse- quently centrifuged at 48,000 g for 30 min. The resulting pellet was then resuspended in ice-cold 50 mM Tris-HCl, pH 7.5. Protein concentration was determined as described by Bradford 7.

Nucleotide regulation of opioid binding [125I]flh-Endorphin, obtained lyophilized, was re-

constituted according to manufacturer's recommen- dations in 0.25% bovine serum albumin (BSA), 5% lactose, 0.2% L-cysteine HC1, 10 mM citric acid and 800 KIU/ml aprotinin. The reconstituted [~25I]fl h- endorphin was aliquoted in 4-ml Nunc polypropy- lene tubes at 1 pCi in 10 pl per tube, and stored at -20 °C until use. One tube (1 pCi) was used for each assay, consisting of 8-12 samples assayed in tripli- cate for a total of 24-36 separate tubes. Just prior to use, unlabeled flh-endorphin in 50 mM Tris-HCl, pH 7.5, 0.2% BSA, and 0.01% bacitracin was added to the 1 pCi of [125I]flh-endorphin to reach the desired

final concentration. Nucleotide regulation of [125I]flh-endorphin bind-

ing was assayed as previously described 31 with some

slight modifications. Briefly, 40 pg of NG108-15 membrane protein or 20 pg of SK-N-SH membrane protein were incubated in a final volume of 0.5 ml with 1.6 nM [125I]flh-endorphin and various nucleo-

tides and/or salts at 25 °C for 60 min in 50 mM Tris-HCl, pH 7.5, containing 0.2% BSA and 0.01% bacitracin. Non-specific binding was measured in the presence of 1 pM flh-endorphin. The incubation was terminated by filtration through Whatman GF/B glass fiber filters that had been soaked in 0.3% polyethylenimine (PEI) for 60 min, using a 48-well Brandel cell harvester. The filters were washed 3 times with 4 ml ice-cold 50 mM Tris-HCl, pH 7.5. Bound radioactivity was determined by counting the filters in a gamma counter.

Nucleotide regulation of the binding of the 3H- labeled ligands was assayed as described above with the following modifications. [3H]DPDPE, at a final concentration of 6 nM, was incubated with 100 pg of NG108-15 membrane protein for 3 h at 25 °C. Non-specific binding was measured by inclusion of 10 pM [D-Ala2,D-LeuS]enkephalin (DADLE) in the

incubation mixture. [3H]DAGO, at a final concen- tration of 1 nM, was incubated with 40 pg of SK-N-SH membrane protein for 60 min. Non- specific binding was measured in the presence of 10 pM DAGO. Filters were not soaked in PEI. Bound radioactivity was determined via liquid scintillation spectrophotometry in 7 ml of Liquiscint scintillation fluid at a counting efficiency of 35-50%.

Statistics All binding assay data which were determined to

be normally distributed were subjected to analysis of variance followed by Dunnett 's multiple range test or Newman-Keuls multiple range test to determine the significance of each individual datum point. Data which were not normally distributed were subjected to Kruskal-Wallis analysis of variance and multiple comparison test. All significance levels reported were the most conservative estimate of significance revealed by post hoc analysis.

Materials [3H]DAGO ([3H]RX783006) (57.5 Ci/mmol) was

purchased from Amersham. [3H]DPDPE ([3H]Mos- berg 6 agonist) (29 Ci/mmol) and [125I]iodotyro- sy127flh-endorphin (2000 Ci/mmol) were obtained from New England Nuclear. GTP and Gpp(NH)p were obtained from PL Biochemicals. All other nucleotides, PEI, and bacitracin were obtained from Sigma. DADLE and flh-endorphin were purchased from Peninsula. EX-CELL 300 medium was ob- tained from J.R. Scientific. Liquiscint scintillation fluid was purchased from National Diagnostics.

RESULTS

To estimate the relative number of p and t~ opioid sites to which [125I]flh-endorphin binds in SK-N-SH cell membranes, displacement of 1.6 nM [125I]fl h- endorphin binding to SK-N-SH cell membranes by DAGO, [D-Ser2,Leu5,Thr6]enkephalin (DSLET), and DPDPE, was examined. [125I]flh-Endorphin binding was most potently inhibited by the p- selective ligand DAGO, as shown in Fig. 1. How- ever, 15 + 1% of bound [125I]flh-endorphin remained in the presence of 5/~M DAGO. Both 6-selective agonists, DPDPE and DSLET, were less potent than DAGO in inhibiting [125I]flh-endorphin binding to SK-N-SH cell membranes. The highly d-selective peptide DPDPE was the least potent displacer over the concentration range of 10 nM to 1 #M peptide. However, 5 nM DPDPE inhibited 15 + 2% of the [125I]flh-endorphin binding. These data indicate that approximately 85% of 1.6 nM [lzsI]flh-endorphin binding was to p sites, while only 15% was to 6 sites, in SK-N-SH cell membranes.

In the absence of cations, [125I]flh-endorphin binding to SK-N-SH cell membranes was not signifi- cantly altered by GTP, as shown in Fig. 2A.

25

However, in the presence of 20 mM NaCI, GTP produced a concentration-dependent inhibition of [125I]flh-endorphin binding. This inhibition was sig- nificantly greater than the inhibition produced by 20 mM NaCI alone over the concentration range of 2-200 btM GTP (P < 0.01 for 5-200 pM; P < 0.05 for 2 pM). Maximal inhibition of [125I]flh-endorphin binding was achieved at 10 #M GTP, where a 22% aecrease in [125I]flh-endorphin binding beyond the decrease produced by NaCI alone was observed. This inhibition, produced by 10 #M GTP, was equivalent to a 34% inhibition of [125I]flh-endorphin binding in the presence of 20 mM NaC1 alone, when the [125I]flh-endorphin bound in the presence of 20 mM NaCI alone was designated as control binding. Half-maximal inhibition of [125I]flh-endorphin bind- ing in the presence of 20 mM NaCI was achieved at 3 ,uM GTP. Similar results were obtained with Gpp(NH)p, which also did not inhibit [125I]fl h- endorphin binding in the absence of cations, as shown in Fig. 2B. In the presence of 20 mM NaC1, Gpp(NH)p produced a significant inhibition of [125I]fl h- endorphin binding in the concentration range of 50-200 #M Gpp(NH)p (P < 0.01). A maximal inhibition of 20% of [125I]flh-endorphin binding beyond the inhibition obtained with NaC1 alone was achieved with 50 #M Gpp(NH)p. This inhibition was equivalent

i v ! i

~,8o r.~

_z 6 0

O n~

Z 0 40 U

2 0 o DSLET • DAGO

DPOPE

0 I i i I - 0 . I I I0 I 0 0 I 0 0 0 IO, OOO

D I S P L A C E R ( n M )

Fig. 1. Displacement of [125I]flh-endorphin binding to SK- N-SH cell membranes by selective opioid agonists. Membranes were incubated at 25 °C for 60 rain with 1.6 nM [~25I~h- endorphin in the presence of varying concentrations of DSLET (O), DAGO (D), or DPDPE (A). Data points represent % control binding + S.E.M. Error bars are not shown where the S.E.M. was smaller than the size of the symbol. All experiments were performed in triplicate and replicated at least 3 times.

26

to a 30% inhibition of [125I]flh-endorphin binding, when the binding in the presence of NaCl was used as control binding. Half-maximal inhibition of [125I]fl h-

endorphin binding by Gpp(NH)p in the presence of NaC1 occurred at 20/~M Gpp(NH)p. Although the maximal inhibition produced by Gpp(NH)p and GTP did not differ significantly, GTP produced greater inhibition than Gpp(NH)p at 5 and 10/~M nucleotide (P < 0.05), in the presence of NaC1. Thus, Gpp(NH)p was less potent than GTP in inhibiting [125I]flh-endor- phin binding to SK-N-SH cell membranes in the presence of 20 mM NaCI.

8° f Z~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

',3

2O O - N o e l

• . I -Noe l

. . . . 2 0 mM N o e l only

I | IO IOO GTP (tiM)

100,

8 0

60

4o O

2O

i

O - N 0 C l

• + N( ICl . . . . 2 0 r a M N o e l only

[ i

I 0 I 0 0

Fig. 2. Effect of GTP and Gpp(NH)p on ['25I]flh-endorphin binding to SK-N-SH cell membranes. Membranes were incu- bated at 25 °C for 60 min with 1.6 nM [125I]flh-endorphin and varying concentrations of GTP (A) or Gpp(NH)p (B) in the presence (Q) or absence (O) of 20 mM NaC1. Data points represent % control binding _+ S.E.M. Error bars are not shown where the S.E.M. was smaller than the size of the symbol• The dashed line indicates % of control binding of [125I]flh-endorphin in the presence of 20 mM NaCI only. All experiments were performed in triplicate and replicated at least 3 times.

[1251]flh-Endorphin binding to NG108-15 cell mem- branes, which contain only b-type opioid receptors s, was also not regulated by GTP in the absence of cations, as shown in Fig. 3A. However, a concen- tration-dependent inhibition of [125I]flh-endorphin binding to NG108-15 cell membranes by GTP was observed in the presence of 20 mM NaCl. The decrease in binding produced by GTP in the pres- ence of NaC! was significantly greater than that obtained with NaC! alone over the concentration

120

i00 q

i 80

6O ~

o 4 0

20

0

O - N o C I

• + N o C l . . . . 2 0 r a M NoCl on ly

I

I0 tOlO GTP (}JM}

ioo (

z 5 8o!

g 4o

2O

~ B -

O - N o C I

• + N o C l

. . . . 2 0 r a m NoCI on ly

I

I0 IOJO Gpp(NHlp (tiM)

Fig. 3. Effect of GTP and Gpp(NH)p on [125I]flh-endorphin binding to NG108-15 cell membranes. Membranes were incubated at 25 °C for 60 min with 1.6 nM [1251]flh-endorphin and varying concentrations of GTP (A) or Gpp(NH)p (B) in the presence (O) or absence (O) of 20 mM NaCI. Data points represent % control binding + S.E.M. Error bars are not shown where the S.E.M. was smaller than the size of the symbol. The dashed line indicates % of control binding of [125I]flh-endorphin in the presence of 20 mM NaC1 only. All experiments were performed in triplicate and replicated at least 4 times.

27

range of 20-200 aM GTP (P < 0.01). Maximal

inhibition was achieved at 100 pM GTP, where a

27% inhibition of [125I]flh-endorphin binding beyond

the inhibition produced by 20 mM NaCl was ob- tained. This inhibition was equivalent to a 41% inhibition of [1251]flh-endorphin binding, when the

binding obtained in the presence of NaCI alone was used as control binding. Half-maximal inhibition of

[125I]flh-endorphin binding in the presence of NaC1 was obtained at 15 pM GTP, a value approximately

5 times greater than the GTP concentration neces-

sary to produce half-maximal inhibition in the

SK-N-SH cell membranes. No significant change in

[125I]flh-endorphin binding to NG108-15 cell mem- branes was produced by Gpp(NH)p in the absence

of cations, as shown in Fig. 3B. Similar to GTP, Gpp(NH)p elicited a concentration-dependent in- hibition of [125I]flh-endorphin binding to NG108-15

cell membranes in the presence of 20 mM NaCi. The

inhibition produced by Gpp(NH)p in the presence of NaCl was significant in the concentration range of

10-200 pM Gpp(NH)p (P < 0.01 for 20-200 ~M; P < 0.05 for 10/~M). Maximal inhibition was achieved

at 50 pM Gpp(NH)p, and was approximately equal

to the maximal inhibition produced by GTP. Half- maximal inhibition by Gpp(NH)p was observed at 12

pM Gpp(NH)p. The inhibition produced by Gpp- (NH)p did not differ significantly from that obtained

with GTP at any concentration of nucleotide. Experiments were conducted to determine the

specificity of cation-induced regulation of [125I]fl h-

endorphin binding by guanine nucleotides. Table I shows the effect of various cations upon [125I]fl h-

endorphin binding to NG108-15 cell membranes in the absence and presence of 100 ~M GTP. All of the

monovalent cations by themselves were approxi- mately equipotent in inhibiting [125I]flh-endorphin

binding to NG108-15 cell membranes. Among the divalent cations, magnesium and calcium were about

20 times more potent than the monovalent cations, while manganese was about 40 times more potent. However, only sodium was significantly effective in promoting inhibition of [125I]flh-endorphin binding to NG108-15 cell membranes by 100 pM GTP (P < 0.01). In the presence of NaC1, GTP inhibited 31% of [125I]flh-endorphin binding, beyond the inhibition obtained with sodium alone.

In contrast to the relative specificity of sodium as

a promoter of GTP-induced regulation of [125I]fl h-

endorphin binding to NG108-15 cell membranes,

Table II shows that significant inhibition of [125I]fl h-

endorphin binding to SK-N-SH cell membranes was

produced by 100 ~M GTP in the presence of sodium (P < 0.01), lithium (P < 0.01), or potassium (P <

0.01). Sodium was most effective among the mono- valent cations in promoting inhibition of [125I]fl h-

endorphin binding by GTP. None of the divalent

cations promoted inhibition of [125I]flh-endorphin binding to SK-N-SH cell membranes by GTP,

though a slight stimulation of [125I]flh-endorphin

binding by GTP was observed in the presence of

MnC12. Sodium and lithium alone were equipotent in inhibiting [125I]flh-endorphin binding to SK-N-SH

cell membranes, while potassium was slightly less

potent. As with the NG108-15 cell membranes,

magnesium and calcium were about 20 times more potent than the monovalent cations by themselves in inhibiting [125I]flh-endorphin binding to SK-N-SH

cell membranes, while manganese was at least 40

times more potent. In order to ascertain the specificity of nucleotide-

induced inhibition of [125I]flh-endorphin binding to

NG108-15 and SK-N-SH cell membranes, various

TABLE I

Effect of cations on regulation of [1251]flh-endorphin binding to NG108-15 cell membranes by G TP

NG108-15 cell membranes, in a final volume of 0.5 ml 50 mM Tris-HCl, pH 7.5, were incubated at 25 °C for 60 min with 1.6 nM [l:sI]flh-endorphin and the indicated concentration of salt in the presence or absence of 100/~M GTP. Data are expressed as mean % inhibition from control binding _+ S.E.M. Parentheses indicate a % increase in binding from control binding. The 'Difference in % inhibition' indicates the difference between the % inhibition in the presence and absence of GTP. In the absence of salt, membranes incubated with 100 pM GTP bound 100.1 _+ 3.0% of [~251]flh-endorphin relative to control binding. All experiments were performed in triplicate and replicated at least 3 times.

Salt (mM) % Inhibition % Inhibition Difference without GTP with l OO ltM in %

GTP inhibition

NaCI (20) 32.6 + 1.2 63.5 + 1.5 30.9 LiCI (20) 47.2 + 3.5 54.1 + 0.2 7.0 KCI (20) 41.1 + 5.5 44.6 + 3.0 3.5 MgCI 2 (l) 43.6 + 1.4 36.8 _ 4.8 (+7.0) CaCI 2 (1) 40.8 + 5.0 35.6 + 4.7 (+5.2) MnCI 2 (0.5) 42.7 + 0.8 44.2 + 2.0 1.5

28

TABLE II

Effect o f cations on regulation o f [1251]flh-endorphin binding to SK-N-SH cell membranes by GTP

SK-N-SH cell membranes, in a final volume of 0.5 ml 50 mM Tris-HCl, pH 7.5, were incubated at 25 °C for 60 min with 1.6 nM [125I]flh-endorphin and the indicated concentration of salt in the presence or absence of 100pM GTP. Data are expressed as mean % inhibition from control binding __ S.E.M. Parentheses indicate a % increase in binding from control binding. The 'Difference in % inhibition' indicates the dif- ference between the % inhibition in the presence and absence of GTP. In the absence of salt, 100pM GTP inhibited [125I]fl h- endorphin binding to SK-N-SH cell membranes by 4.4 + 0.5%. All experiments were performed in triplicate and replicated at least 3 times.

Salt (mM) % Inhibition % Inhibition Difference without GTP with IO0 t~M in %

GTP inhibition

NaCI (20) 36.1 _+ 0.7 58.5 + 1.2 22.4 LiC1 (20) 38.3 _ 1.3 55.0 _ 1.3 16.7 KCI (20) 25.9 + 0.7 36.2 + 1.2 10.3 MgCI2 (1) 30 .2+ 1.1 30 .6+0 .7 0.4 CaCI2 (1) 33 .7+ 1.0 30.0 + 1.8 (+3.7) MnCI~ (0.5) 49.2 + 0.7 39.6 + 0.7 (+9.6)

nucleotides were substituted for GTP in the absence and presence of 20 mM NaC1. As shown in Table III , no significant change in [125I]flh-endorphin binding to NG108-15 cell membranes was observed with any nucleotide in the absence of cations. In the presence of 20 mM NaCI, however, 100 ~M GDP produced a 25% decrease in [125I]flh-endorphin binding to NG- 108-15 cell membranes. This inhibition was not significantly different from that produced by GTP in

the presence of NaCI. While a slight stimulation by GMP of [125I]flh-endorphin binding to NG108-15 cell membranes was observed in the presence of 20 mM NaC1, none of the non-guanine nucleotides pro- duced any significant change in [125I]flh-endorphin binding. Similar results were obtained when the effects of the various nucleotides on [125I]flh-endor- phin binding to SK-N-SH cell membranes were examined, as shown in Table IV. None of the nucleotides altered [125I]flh-endorphin binding in the absence of cations. Inclusion of 100/~M GDP in the presence of NaC1 resulted in a 25% decrease in [125I]flh-endorphin binding beyond the decrease ob- tained with NaCI alone. The inhibition produced by

TABLE III

Effect o f nucleotides on [1251]flh-endorphin binding to NG108- 15 cell membranes

NG108-15 cell membranes, in a final volume of 0.5 ml 50 mM Tris-HCI, pH 7.5, were incubated at 25 °C for 60 min with 1.6 nM [lzsI]flh-endorphin and the indicated concentration of nucleotide in the presence or absence of 20 mM NaCI. Data are expressed as mean % inhibition from control binding + S.E.M. Parentheses indicate a % increase in binding from control binding. The 'Nucleotide effect with 20 mM NaCI' indicates the % inhibition in the presence of NaCI and nucleotide minus the % inhibition in the presence of NaCI alone. In the absence of nucleotide, 20 mM NaCI inhibited [a25I]flh-endorphin binding to NG108-15 cell membranes by 32.6 + 1.2%. All experiments were performed in triplicate and replicated at least 4 times.

Nucleotide % Inhibition % Inhibition Nucleotide (raM) without NaCI with 20 mM effect with

NaCI 20raM NaCI

GTP (100) (+0.1 + 3.0) 63.5 + 1.5 30.9 GDP (100) 12.3 + 3.3 57.9 + 2.0 25.3 GMP (100) (+2.9 + 2.4) 20.7 + 2.7 (+11.9) CTP (100) 2.0 + 2.5 34.1 + 2.6 1.5 ATP (100) 6.7 + 3.6 37.1 + 3.4 4.5 UTP (100) 5 . 2 + 4 . 4 37 .1+1 .4 4.5 TTP (100) 3.6_+2.1 32 .5+1 .6 (+0.1)

TABLE IV

Effect o f nucleotides on [~251]flh-endorphin binding to SK-N-SH cell membranes

SK-N-SH celt membranes, in a final volume of 0.5 ml 50 mM Tris-HCl, pH 7.5, were incubated at 25 °C for 60 min with 1.6 nM [125I]flh-endorphin and the indicated concentration of nucleo- tide in the presence or absence of 20 mM NaCI. Data are expressed as mean % inhibition from control binding + S.E.M. Parentheses indicate a % increase in binding from control binding. In the absence of nucleotide, 20 mM NaC1 inhibited [125I]fl h- endorphin binding to SK-N-SH cell membranes by 36.1 + 0.7%. The 'Nucleotide effect with 20 mM NaCI' indicates the % inhibition in the presence of NaC1 and nucleotide minus the % inhibition in the presence of NaCI alone. All experiments were performed in triplicate and replicated at least 3 times.

Nucleotide % Inhibition % Inhibition Nucleotide (I~M) without NaCI with 20 mM effect with

NaCl 20 mM NaCI

GTP (100) 4.4 + 0.5 58.5 + 1.2 22.4 GDP (100) 4.6 + 2.8 60.9 + 0.8 24.8 GMP (100) 1.3 + 0.5 38.4 +__ 1.2 2.4 CTP (100) 0 .4+ 1.3 37.6 + 1.5 1.6 ATP (100) (+3.4 + 1.4) 35.7 + 2.1 (+0.4) UTP (100) (+0.2 + 2.1) 41.2 + 1.6 5.2 TFP (100) 1.1 + 1.9 41.1 + 2.2 5.0

TABLE V

Effects of guanine nucleotides on [3H]DPDPE binding to NG108-15 and [3H]DAGO binding to SK-N-SH cell mem- branes

NG108-15 cell membranes, in a final volume of 0.5 50 mM Tris-HCl, pH 7.5, were incubated at 25 °C for 3 h with 6 nM [3H]DPDPE and the indicated nucleotide. SK-N-SH cell membranes were incubated at 25 °C for 60 rain with 1 nM [3H]DAGO and the indicated nucleotide. Data are expressed as mean % inhibition from control binding + S.E.M. Parentheses indicate a % increase in binding from control binding. All experiments were performed in triplicate and replicated at least 3 times.

Nucleotide (pM) % Inhibition by nucleotide

NG108-15 SK-N-SH

GMP (100) (+0.7 _+ 2.7) 2.8 _+ 2.8 GDP (100) 26.2 _+ 4.9 32.8 _+ 2.6 GTP (100) 22.1 _+ 5.6 39.8 + 2.4 Gpp(NH)p (100) 24.6 __ 4.4 25.1 + 0.9

100 pM GDP in the presence of NaCI did not differ significantly from that obtained with GTP. All other nucleotides tested did not affect [125I]flh-endorphin binding to SK-N-SH cell membranes in the presence of 20 mM NaC1.

The regulation of [3H]DPDPE binding to NG108- 15 cell membranes and [3H]DAGO binding to SK-N-SH cell membranes by guanine nucleotides was tested in the absence of cations. As shown in Table V, 100 pM GDP, GTP, and Gpp(NH)p inhibited [3H]DPDPE binding to NG108-15 cell membranes and [3H]DAGO binding to SK-N-SH cell membranes in the absence of cations (P < 0.01), while 100 pM GMP had no effect.

DISCUSSION

In this report, we have demonstrated that [125I]fl h- endorphin binding to NG108-15 and SK-N-SH cell membranes was inhibited by guanine nucleotides in the presence of certain monovalent cations, but not in their absence. This is in contrast to the binding of the 6- andp-selective enkephalin analogues, [3H]DP- DPE and [3H]DAGO, respectively, which was in- hibited by guanine nucleotides in the absence of cations. These results are in agreement with our previous report on the regulation of [125I]flh-endor- phin binding to rat brain membranes by guanine nucleotides 31.

29

The major difference between the NG108-15 and SK-N-SH cell membranes with regard to guanine

nucleotide-induced regulation of [125I]flh-endorphin binding was the specificity of the cation requirement. Regulation by GTP of [~25I]fln-endorphin binding to the p receptor-enriched SK-N-SH cell membranes occurred in the presence of all of the monovalent cations tested, with their order of potency being: sodium > lithium > potassium. This monovalent cation requirement is identical to that previously demonstrated for GTP-induced regulation of [1251]-

flh-endorphin binding to rat brain membranes, with the same order of potency 31. In the NG108-15 cell

membranes, however, only the presence of sodium resulted in inhibition of [125I]flh-endorphin binding by GTP. Though a small amount of inhibition of [125I]fln-endorphin binding by GTP was observed with lithium, this inhibition was not significantly different from that produced by the salt alone. Thus, guanine nucleotide-induced regulation of [125I]fl h-

endorphin binding to 6 receptors in NG108-15 cell membranes has a more specific requirement for sodium than does this effect in either SK-N-SH cell or rat brain membranes.

It is unclear whether the greater specificity of the cation requirement for regulation of [125I]fln-endor- phin binding to NGI08-15 cell membranes results from a mechanism of action of the monovalent cations that differs between the opioid receptors in NG108-15 cell membranes and those in the SK- N-SH cell and rat brain membranes. Puttfarcken et al. 29 have shown that the effect of sodium on the binding of opioid agonists to NG108-15 cells was to decrease the apparent Bma x value of 6 receptors by converting a portion of these receptors to a very low affinity state. This sodium effect was concurrent with a sodium-dependent increase in the maximal inhib- itory effect of D A D L E and fl-endorphin upon adenylate cyclase activity in the NG108-15 cell. However, in the /~ receptor-containing pituitary

tumour cell line 7315c, sodium increased the IC5o value of DADLE and morphine for inhibition of adenylate cyclase activity. This action of sodium was consistent with the decrease in agonist affinity of p receptors observed in the presence of sodium. Thus, there is a distinct possibility that the sodium-specific requirement of GTP-induced inhibition of [1251]fl h- endorphin binding to NG108-15 cell membranes is

30

related to the sodium requirement for 6 receptor-N i coupling in NG108-15 cells. Indeed, it has been shown that opioid-induced stimulation of low K m GTPase activity, presumably associated with activa- tion of N i, requires both GTP and sodium in NG108-15 cells 21. Blume et al. 6 reported that sodium is required for inhibition of adenylate cyclase activity by the opioid agonist DADLEamide, with an ap- parent K m for NaC1 of 20 mM. However, lithium and potassium were shown to mimic this sodium effect, with the order of potency being: sodium/> lithium > potassium. This order of potency corresponds well with the order of potency of these ions in promoting GTP-induced inhibition of [125I]flh-endorphin bind- ing to SK-N-SH cell and rat brain membranes. Studies of the effect of sodium on opioid-induced inhibition of adenylate cyclase in rat brain have revealed that sodium enhanced the effect of the opioid 3'13, produced no change in the effect of the opioid ~9, or reduced the inhibition produced by opiate alkaloids, and increased or had no effect upon that produced by opioid peptides 26. Furthermore, it has been reported that sodium decreased the po- tency of opioid agonists in inhibiting adenylate cyclase activity in NG108-15 cells or cell membranes, but increased the agonist potency for inhibition of adenylate cyclase activity in N18TG2 cells 25. Thus, the effect of sodium on opioid receptor-second messenger coupling remains controversial, and seems to be dependent upon both the system and the agonist which is investigated. This latter condition is consistent with the finding that there was a specific monovalent cation requirement for regulation of [~2sI]flh-endorphin binding by guanine nucleotides, while there was no such requirement for similar regulation of the binding of [3H]DAGO or [3H]- DPDPE. This may reflect a more complex interac- tion of fl-endorphin with opioid receptors than that of the smaller opioid peptides or opiate alkaloids.

Another difference between GTP-induced regu- lation of [~25I]flh-endorphin binding to NG108-15 and SK-N-SH cell membranes was the relative potency of GTP and Gpp(NH)p in the presence of sodium. In the SK-N-SH cell membranes, half- maximal inhibition of [125I]flh-endorphin binding in the presence of sodium was achieved with 3 pM GTP, while 20 pM Gpp(NH)p was required to

produce half-maximal inhibition under these condi- tions. In contrast, the concentration values for half-maximal inhibition of [~25I]flh-endorphin bind- ing to NG108-15 cell membranes by GTP or GPP- (NH)p in the presence of sodium were 15 and 12/aM, respectively. In rat brain membranes, half-maximal inhibition of [125I]flh-endorphin binding in the pres- ence of sodium was obtained with 5/~M GTP 31, a

value similar to that observed for GTP in SK-N-SH cell membranes. The value for half-maximal Gpp(NH)p-induced inhibition of [125I]flh-endorphin binding to rat brain membranes in the presence of sodium was 1 ,uM 31. This value is much lower than that observed with either NG108-15 or SK-N-SH cell membranes in this study. The reasons for the discrepancies in nucleotide potency among the var- ious sources of opioid receptor are presently unclear, though further investigation may reveal whether these differences are dependent on the relative number of p and 6 opioid receptors present in the tissue.

In conclusion, we have shown that inhibition of [125I]/3h-endorphin binding to NG108-15 and SK- N-SH cell membranes by guanine nucleotides pos- sesses a specific monovalent cation requirement. This requirement may be a consequence of the effect of these cations on opioid receptor-N i coupling combined with a greater complexity of the interac- tion of fl-endorphin with opioid receptors than that of the smaller opioid peptides and opiate alkaloids. The characteristics of GTP-induced regulation of [125I]flh-endorphin binding to SK-N-SH cell mem- branes closely parallel those of the regulation of [125I]flh-endorphin binding to rat brain membranes by GTP. The regulation of [125I]flh-endorphin bind- ing to NG108-15 cell membranes by guanine nucleo- tides displays properties which differ from those observed in rat brain membranes. These results may indicate that /~ opioid receptors are a greater component of guanine nucleotide-sensitive fl-endor- phin binding than 6 opioid receptors in rat brain.

ACKNOWLEDGEMENTS

This work was supported by USPHS Grants DA05319 and DA03742 from the National Institute on Drug Abuse.

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