Characterization of binding sites for [3H]-DTG, a selective sigma receptor ligand, in the sheep...

Vol. 171, No. 2, 1990

September 14, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 8~5881

CHARACTERIZATION OF BINDING SITES FOR L3Hl-DTG, A SELECTIVE SIGMA RECEPTOR LIGAND, IN THE SHEEP PINE&L GLAND

Pedro Abreu and David Sugden

Biomedical Science Division, King's College London, Campden Hill Road,

Kensington, London W8 7AH.U.K.

Received July 25, 1990

Sp5cific binding sites for 13H)-1,3 di-ortho-tolylguanidine (I HI-DTG), a selective radiolabeled sigma receptor ligand, were detected an 4 characterized in sheep pineal gland membranes. The binding of [ HI-DTG with rate

to sheep pineal membranes was rapid and reversibly

-la constant for

.min association (K+,) at 25 C of-y.0052 nM

and rate constant for dissociation (K (K /K+,) of 9.9 nM. S t

) 0.0515 min , gfving a

%nds -' a uration studies dLmonstrated that I HI-DTG

to a single class of sites with an affinity constant 27 + 3.4 nM, and a total binding capacity (B

(Kd) of

pmol/mg protein. Competition ) of 1.39 + 0.03

experiments showed the relative order of potency of compounds for inhibition of

g!%t [ HI-DTG binding to

sheep pineal membranes was as follows: trifluoperazine = DTG> haloperidol> pentazocine> (+)-3-PPP > (+/-)SKF 10,047. Some steroids (testosterone, progesterone, deoxycorticosterone) previously reported to bAnd to the sigma site in brain membranes were very weak inhibitors

[ HI-DTG binding in the present study. The ?H 1 -DTC

results indicate that 1 binding sites having the characteristics of sigma receptors

are present in sheep pineal gland. The physiological importance of these sites in regulating the synthesis of the pineal hormone melatonin awaits further study. @1990 Academic Press, Inc.

In the pineal gland, noradrenaline released at night from

sympathetic fibres innervating the gland, activates both 5-

and

B-adrenoceptors to induce serotonin N-acetyltransferase (sNAT)

activity, the rate-limiting enzyme in the synthesis of the pineal

hormone melatonin (1). In addition to these adrenergic receptors

various other receptors have been described in this tissue. Receptors

for vasoactive intestinal polypeptide (VIP) are present and their

activation elevates intracellular cyclic AMP leading to an induction

of sNAT activity (2,3). Other receptors have been described using

techniques including dopaminergic D2 (4),adenosine

(6) and benzodiazepine receptors (7). However the

radioligand binding

A2 (5), muscarinic

OOU6-291X/90 $1.50

875

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 171, No. 2, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

importance of these sites in regulating pineal physiology is not

known.

The present study identifies and characterizes binding sites in the

sheep pineal gland for [3H1-l,3-di-o-tolylguanidine (t3~1-~T~), a

selective ligand for sigma receptors (8). The site selectively

labelled by L3Hl-DTG is the high affinity, naloxone-insensitive site

previously identified in brain using the synthetic sigma opiate drug

[3H]-(+)-N-allylnormetazocine (SKF 10,047) (9) and L3Hl-(+)-3-

(3-hydroxyphenyl)-N-(l-propyl)piperidine ((+)-3-PPP) (10). The study

was prompted by recent autoradiographic data describing a particularly

high density of sigma receptors in the rat pineal gland in comparison

with rat brain (11) and is part of a continuing effort to identify the

receptor sites present on pineal ocytes and to understand their role in

regulating melatonin synthesis.

MATERIALS AND METHODS

1,3-Di-o-tolylguanidine,di-[p-ring-3Hl-, (L3~l-~~~, 52.3 Ci/mmol) was purchased from DuPont (Stevenage, Hertfordshire, U.K.). Drugs were obtained from Sigma Chemical Company (Poole, Dorset, U.K.) and Research Biochemicals Inc. (Natick, MA, U.S.A.).

Tissue Preparation: Adult sheep pineal glands were generously provided by Dr

P.J.Morgan, Rowett Research Institute, Aberdeen, U.K. Glands were stored at -7oOc until used. Five pineals were homogenized in 10 volumes of ice-cold tris HCl buffer (50 mM, pH 7.40) containing 1 m phenylmethylsulfonyl fluoride (PMSF), 50 ug/ml leupeptin and 1 mM EGTA. The homogenate was centrifuged at 100,000 x g for 60 min. at 4Oc. Pellets (crude membranes) were washed twice with homogenization buffer and resuspended at a concentration of 1.67 mg/ml and stored at -7Q'C until used.

Binding Assay: Membranes (30 ~1, 50 1-19) were incubated with 50 ul of [3~1-~~~

in the absence (total binding) or presence (non specific binding) of 10 uM DTG (final concentration) in a total volume of 100 ~1. Kinetic and competition experiments used a concentration of 25 nM L3~1-D~c, and saturation experiments a range of concentrations between 0.625 and 160 Ml. After incubation for 30 min at 25OC in a water bath, 2 ml of ice-cold tris HCl was added and the contents of each tube rapidly filtered under vacuum through GF/C glass-fiber filters (Whatman, Maidstone, U.K.). Each filter was washed twice with 5 ml of ice-cold tris HCl and the radioactivity retained by the filter measured after addition of 4 ml of scintillation fluid (OptiPhase Safe, LKB, UK) on a LKB scintillation spectrometer (model 1219) at a counting efficiency of 55-60%.

876


The protein concentration in the membrane preparation was determined with a dye-binding method (12) using bovine serum albumin as a standard.

Data analysis:. Data from kinetic experiments were analyzed using pseudo

first-order equations to estimate the association rate constant (K ) and the dissociation rate constant (K ). Data from saturat!oL experiments were analyzed using ENZFITTAR (13).

IC50 values were determined from curves of inhibition of binding against drug concentration fitted using a weighted logit-log equation. Between 9 and 12 inhibitor concentrations were used. K. values + the standard error of the estimate for each inhibitoriwere then-determined from IC 5. values using the Cheng-Prusoff equation (14).

RESULTS AND DISCUSSION

Figure 1 shows that the association of L3H1-DTG to sheep pineal

membranes proceeded rapidly at 25OC reaching a plateau after

approximately 20 min. Dissociation was initiated after 70 minutes by

addition of DTG (10 )JM). Kinetic analysis of data from two experiments

gave a dissociation rate constant (K-,) of 0.0515 + 0.007 -1 min , and

an association rate constant (K+l ) of 0.00520 + 0.0005 nM-'.min-'. The

Kd (Kml,/K+,) value calculated from these constants was 9.90 + 1.6 nM.

Total and non specific binding of L3H1-DTG increased over the

range of concentrations (0.625-160 nM) of L381-~~c used, as shown for

a representative experiment in Figure 2. Specific binding accounted

for 82-86% of total binding. Scatchard analysis of the specific

binding data yielded a linear plot suggesting a single class of

0.6 - DTGt 1 OyM)

I/l.

\ *--a

--v

0 20 40 60 80 100 120

TIME (mid

Figure 1. Kinetics of association and dissociation of L3H1-DTG to sheep pineal membranes. s embrane suspension (50 !Jg protein/tube) was incubated with 25 nM [ HI-DTG at 25OC. Cold DTG (10 PM) was added at the arrow. The specific binding data shown is from a typical experiment. Each point represents the mean of duplicate determinations.

877


binding site for f3H1-DTG (Figure 3). Analysis of 3 independent

experiments gave a mean (2 S.E.M.) Kd of 27.0 + 1.0 nM, a maximal

number of binding sites (Bmax ) 1.39 + 0.03 pmol L3Hl-DTG/mg protein,

and a Hill coefficient (nH) 0.99 + 0.04. The Kd value from Scatchard

analysis and that calculated from the kinetic constants were similar,

and in close aggreement with the Kd (28 nM) reported for i3Hl-DTG

binding to guinea pig brain membranes (8). Binding of [‘)HI-DTG to

sheep pineal membranes was not inhibited by 1 mM GTP (data not shown).

In order to verify that 3 the [ HI-DTG binding site in pineal

membranes was a sigma site the ability of a number of drugs to compete

for ['HI-DTG binding was studied. The rank order of potency of drugs

for inhibiting the binding of [3~1-~~~ (25 nM) to sheep pineal

membranes was: trifluoperazine = DTG > haloperidol > pentazocine >

spiperone> imipramine> propranolol >desimipramine = (+)-3-PPP >

(+/-)SKF 10,047. The potency of these compounds in sheep pineal

membranes was similar to that reported for guinea pig brain membranes

(8).

_ 60.0 2.5

I

Total Binding

0 ? 2.0 - 60.0 .

gz ii * a7

r p 1.5 Specific Binding t- m .

CJ ! f 40.0 & 2 1.0

+ 5 0.5

* ; 20.0 .

Non Specific Binding z 0.0 -

0.0 0 500 1000 1600

0 0 100 200

2 0 3 BOUND 3H-DTG(nM) (fmol/mg. prot.)

Figure 2. Saturation isotherm of r3H1-DTG in sh$ep pineal membranes. Membranes (50 I.rg) were incubated at 25OC with [ HI-DTG (0.625-160 nM) for 30 minutes. Each value is the mean of duplicate determinations. A representative experiment is shown. Non specific binding (V) was determined in the presence of 10 PM DTG. Specific binding (0) was defined as total binding (0) minus non specific binding.

Figure 3. Scatchard plot of L3B1-DTG to sheep pineal membranes. A representative experiment is shown. For further details see legend to Figure 2 and Materials and Methods. Each value is the mean of duplicate determinations.

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In our experiments haloperidol was slightly less potent than DTG.

However, the reduced metabolite of haloperidol (metabolite II),

recently shown to have a high affinity for sigma receptors but a much

lower affinity for the dopamine D 2

receptor (151, inhibited L3H]-DTG

binding to sheep pineal membranes (Table 1). The haloperidol

metabolite formed by oxidative N-dealkylation (metabolite I) was a

much weaker inhibitor (Table 1, 15).

Testosterone, progesterone and desoxycorticosterone, steroids

previously reported to have some affinity for the sigma site in brain

membranes and suggested to be potential endogenous ligands for the

sigma receptor (161, were very weak inhibitors of L3~1-~~c binding in

Table 1. Inhibition of specific [3Hl-DTG binding to sheep

pineal gland membranes

Compound Ki (nM)

Trifluoperazine 10.4 + 4.4 -

DTG 12.5 + 0.6 -

Haloperidol 49.2 2 3.4

Pentazocine 155.0 + 14.0 -

Haloperidol metabolite II 186.6 + 39.8 -

Spiperone 206.7 + 29.0

Imipramine 314.0 + 41.0

Propranolol 345.2 + 17.8

Desimipramine 790.0 2 161.0

(+)-3-PPP 812.0 + 34.0

(+/-JSKF 10,047 1017.4 2 290.9

Haloperidol metabolite I 1253.2 5 201.6

-

The concentration of incubation (25OC.

[3~1-~~~ used was 35 nM. The 30 min) was started by addition of [ HI-DTG and

terminated by filtration as described in Material and Methods. K. values (mean + the standard error of the estimate) were calculate a using the Chenq-Prusoff equation (14) from IC values obtained using a weighted logit-log analysis of competitio?' data. The following compounds caused no significant displacement at 10 UM: 5-HT. GABA, melatonin, phenylephr ine, isoprenaline, noradrenaline, dopamine, (+)pindolol, atenolol, yohimbine, practolol, salbutamol, methoxamine.

The K. values for clonidine, testosterone and desoxykorticosterone were >

progesterone, 10 PM.

879


the present experiments (Ki> 10 UM). Interestingly, these steroids

have been shown to inhibit B-adrenergic induction of sNAT activity

in the rat pineal gland in culture by an action at a site after cyclic

AMP generation (17) at concentrations similar to those reported to

inhibit binding of sigma receptor ligands in brain membranes (16). The

3 very weak inhibition of [ HI-DTG binding in sheep pineal membranes in

the present experiments suggests that it is unlikely that these

steroids inhibit b-adrenergic induction of sNAT activity by an action

on the sigma receptor.

The results confirm, and extend, a recent report (11) using an

autoradiographic method that sigma binding sites are highly

concentrated in the rat pineal gland. Our own preliminary work has

also identified specific L3~l-~TG binding sites in rat pineal

membranes (unpublished). It is not known if these binding sites are

located presynaptically in the sympathetic terminals which innervate

the pineal gland or postsynaptically on pinealocytes themselves. The

role of these binding sites, if any, in regulating pineal physiology

has yet to be determined. Interestingly, sigma liqands have been shown

to attenuate phosphatidylinositol turnover in rat brain synaptosomes

(18); such an action on pinealocytes might be expected to lead to an

inhibition of melatonin synthesis as an a1-

adrenergic activation of

phosphatidylinositol hydrolysis is involved in inducing sNAT activity

(1). In addition sigma liqands have been reported to increase

electrically evoked noradrenaline release from vas deferens (19). If

this effect was to occur in the pineal gland then adrenergic induction

of melatonin synthesis would be expected to be amplified by activation

of sigma receptors. The pineal gland may prove to be a useful model

system in which to study the intracellular signalling systems coupled

to sigma receptors.

ACKNOWLEDGMENTS

We would like to thank Dr P.J.Morqan for supplying sheep pineal glands and Nelson Chong for his help in preparing pineal membranes. P.A. was

880


supported by a postdoctoral visiting fellowship from the Government of the Canary Islands, Spain. The financial support of the Royal Society is gratefully acknowledged. DS is a Royal Society 1983 University Research Fellow.

REFERENCES

(1) Sugden,D. (1989) Experientia 3, 922-932. (2) Kaku,K., Inoue,Y., Matsutani,A., 0kubu.M.. Hatoo,K., Kaneki,T.,

and Yanaihara,N. (1983) Biomed.Res. 4, 321-328. (3) Yuwiler,A. (1983) J.Neurochem. 41, 146-153. (4) Simonneaux,V. Murrin,L.C. and Ebadi,M. (1990) J.Pharmacol.Exp.

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J.Neurochem. 53, 733-737. (6) Taylor,R.L., Albuquerque,M.L. and Burt,D.R. (1980) Life Sci. 2,

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(7) Matthew,E., Parfitt,A.G., Sugden,D., Engelhardt,D.L., Zimmerman, E.A. and Klein,D.C. (1984) J.Pharmacol.Exp.Ther. 228, 434-438.

(8) Weber, E., Sonders, M., Quarum, M., McLean, S., Pou, S. and Keana,J.F.W. (1986) Proc. Natl. Acad. Sci. U.S.A 83, 8784-8788.

(9) Largent,B.L., Gund1ach.A.L. and Snyder,S.H. (1986) J.Pharmacol. Exp.Ther. 238, 739-748.

(10) Gundlach,A.L., Largent,B.L. and Snyder,S.H. (1986) J.Neuroscience 5, 1757-1770.

(11) Jansen, K.L.R., Dragunow, M. and Faull, R.L.M. (1990) Brain Res. 507 --I 158-160.

(12) Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. (13) Leatherbarrow, R.J. (1987) Elsevier-Biosoft, Cambridge, UK. (14) Cheng, Y. and Prusoff, W.H. (1973) Biochem. Pharmacol. 2,

3099-3108. (15) Bowen,W.D., Moses,E.L., Tolentin0,P.J. and Walker,J.M. (1990)

Eur.J.Pharmacol. 177, 111-118. (16) Su, T.P., London, E-D., Jaffe, J.H. (1988) Science 240, 219-230. (17) Yuwiler,A. (1989) J.Neurochem. z, 46-53. (18) Bowen,W.D., Kirschner,A.H., Newman,A.H. and Rice,K.C. (1988)

Eur.J.Pharmaco1.x. 399-400. (19) Campbell,B.G., Bobker,D.H., Les1ie.F.M.. Mefford,I.N. and

Weber,E. (1987) Eur.J.Pharmacol. 138, 447-448.

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Characterization of binding sites for [3H]-DTG, a selective sigma receptor ligand, in the sheep...

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