hi-opmnz .htrnul of Piwniacology, 505 ( 1YYI ) I-6 0 1YYI Elsevier Science Publishers B.V. All rights reserved OOl4-2YYY/Y1/$03.50

EJP 52135

Arie H. Mulder, George Wardeh, Fransois Hogenboom, Wieslaw Kazmierski I, Victor J. and Anton N.M. Schoffelmeer

Hruby

I

Received h June 1901, revised MS received 31 July IYYI. acceplrd 27 August IYYI

The opioid receptor antagonist propertics of four conformationally conslraincd cyclic octapcptidc analogucs of somatosIaIin were investigated using in vitro functional paradigms of cc-, S- and K-opioid rcccplors in Ihc rat brain. The analogucs cxamincd were D-Phe-Cys-Tyr-D-Trp-Om-Thr-Pen-Thr-NH z (CTOP9, D-Phc-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH 1 (CTAP), D-TicCTOP (TCTOP9 and D-Tic-CTAP (TCTAP). Activation of p-rcccptors by the cnkcphalin analoguc Tyr-D-Ala-Gly-(NMo)PhcGly.01 (DACO9 inhibited Ihc (clcctrically cvokcd9 rclcasc of [3Hlnoradrcnalinc (NA9 from supcrfuscd cortical slices and this inhibitory cffcct was antagonized in a compctitivc fashion by all of the octapcptidcs Icstcd (PA, values: CTOP and CTAP 7.940, TCTOP and TCTAP 8.7-8.89. Sclcctivc activation of K-opioid receptors by Ihc cyclohcxylhcrizcncacctamidc UhYSY3 (0.02 PM) inhibited (by 40-45%) the rclcasc of [‘Hldopaminc (DA9 from SIriatal slices. whcrcas sclcctivc activation of &opioid rcccptors by [D-Scr’(O-t-buIy19,Lcu51cnkcphalyl-Thr” (DSTBULET; (1.1 pM9 c;luscd an inhibiIion (by 3X-W%) of striatal [‘4C]acctylcholinc (ACh9 rclcasc. However, thcsc inhibitory cffccIs wcrc noI affccIcd by any of thu OctilpcpIidCh in conccntralions that caused full anlagonism trf Ihc inhibitory cffcct (5.5~05% 1 of 0.1 PM DAGO on corIic;d [ ‘H]NA rclc;lsc. Thus, the cyclic octapcrtids somatostatin analogucs CTOP, CT,;P, TCTOP and TC’TAP arc potent and highly x+cIivc anlagonixts ilt Ihc y-opioid receptors mediating prcsynaptic inhihition of NA rclcasc in the brain. The p-rcccptor affiniIy ol the mosl poIcnl rll Ihcsc antagonists, TCTOP and TCTAP, appears to hc similar to that of nalwwnc hul thcsc irnl;lg(~nisls IWVC ;I much grcatcr selectivity than th’c latter.

Somatostatin

analogucs; Opioid rcccptor antigonisth;

Opioid receptors; NcurotransmiIIcr

1. lntrodurtion Several years ago the tctradccapcptidc somatostatin (SS-14) was shown to display some affinity for opioid receptors, in spite of the apparent lack of structural similarity of SS-I4 to the endogcnous opioid peptidcs or the opiate alkaloids (Tcrenius, lW6). Later, iIruby and coworkers synthesized various conformationally constrained cyclic octapcptide somatostatin analogucs, containing penicillamine (Pen: &p-dimcthylcystcincJ, which appcarcd to possess greatly enhanced affinity for opioid receptors combined with strongly rcduccd somatostatin-like activity, as dctcrmincd by radioligand binding assays in brain mcmbrancs (Pclton ct al., 10X5, lc)M; Gulya ct al., IWW. Thus far, the most intcrcsting

rclcasc; Prcsynaptic inhibition

mcmhcrs of this scrics of octapcptidcs appear to he C’TP (D-l’hc-Cys=?‘yr-D-‘I’rp-Lys-‘l’hr-l’cn-~~~~r-NH2 1, its derivatives CTOP (D-Phc-Cys-Tyr-D-Trp-Orn-ThrPen-Thr-NH :) and CTAP (D-Phc-Cys-Tyr-D-Trp-ArgThr-Pen-Thr-NH ?9, in which the Lys rcsiduc of CTP is rcplaccd by Orn or Arg, rcspcctivcly, and the more rcccntly dcsigncd analogucs TCTOP (I>-Tic-C‘TOP9 and TCTAP (D-Tic-CTAPI (Kazmicrski ct ai., lWH9, obtained by substituting D-*ctrahydroist,quinolinc carboxylic acid for D-Phc in CTOP or CTAP (fig 19. Functional studies in vitro using pcriphcral tissue preparations, such as the clcctrically stimulated isolatcd guinea-pig ileum and mouse vas dcfcrcns. rcvcaicd that thcsc pcptidcs arc quite potent opioid rcccptor antagonists with a high dcgrcc of sclcctivity towards the p-opioid rcccptor type (Shook ct al., 1987; Kasmicrski ct A., IWX). This high p-sclcctivity has ak-~) been I’6)und in r;IdioligiInd binding ilSSilyS ill brilin mcmbrancs, whc~ C’I’I appcarcd to have about 300fold hi&r ilffinity for p- lh:ln for fi-opid rcrcpto9

. l)r

II-l’hr

. I‘>\

Il.Phr

- C‘>s - l)r

D-Tic - C?%

II-Tic

. I).lbp

- Ljs

. Thr

- Pen - Thr.NH,

. I).lbp . Om- Thr - Pen- Thr-NH,

. nr - I>-‘Rp

. Om

- Thr

. Pen - Thr-NH,

. C’?c- ljr . WRp - Arg. Thr . ICn. Thr.WI,

CTP

(‘TOP

TCTOP

‘T(‘TW

Fig. I. Amino ;u$l squcnct’s of CTP and the related cyclic wmatostatin ;malogues rwmined in the present study. Orn = urnithinc: Pen = prnicillaminr (~.~-dimcthglcysteine): D-Tic = D-tctrilhytlrtrisoquinolins carhoxylic acid.

binding sites, CTOP 1300-fold. CTAP 2500-fold. ,TCTAP 1000-fold and TCTOP even more than IOOOO-fold (Kazmicrski et al., 1988). In the present study WC‘ examined the apparent affinity and sclcctivity of CTOP. CTAP. TCTOP and TCTAP as antagonists for opioid receptors in the brain, using inhibition of neurotransmittcr release as a functional paradigm in vitro for these receptors. Studies with rat brain slices have shown that selective activation of each of the major opioid receptor types (i.e. I?, K, p) may result in selective inhibition oi the dcpolarization-induced release of a parlicular ncurotransmittcr (Muldcr et al., 1988; Illcs, 19X9). We studied the p-receptors mediating prcsynaptic inhibi!ion of the (electrically evoked) release of [‘Hlnoradrenaline (NA) from cortical slices (Wcrling e! al., 1987; Muldcr ct al., 19X8; Schoffelmcer et al., 19X8) and the K- and &rcceptors mediating inhibition of [.7H]dopaminc (DA) and [‘JC]acctylcholinc (ACh) rclcasc from striatal slices, respectively (Mulder et al., 19114. 198X; Schoffclmecr et al., 1988) Tyr-D-Ala-Gly-(NMcIPhe-Gly-ol (DAGO). [D-Ser’(O-t-butyi),Leu’]enkephalyl-Thr” (DSTBULET) and the cyclohexylbcnzcncacctamide derivative UhYSY3 were used as highly selective agonists for p-, S- and K-opioid receptors, respectively (Mulder et al., 1989, 1991; Clark et al., 1088; Gacel et al., 1988; De Vries et al., 1989).

2. Materials and methods 2.1. Ptqmmtiott

attd itrcrrbation

of bruitr

slices

Male Wistar rats (l40-180 g body weight! wcrc decapitated and the brains were rapidly removed. Ncocortical and neostriatal slices (approximately (1.3 x 0.3 x 2 mm) were prepared, labellcd and supcrfuscd cssentially as described previously (Schoffelmecr et al., 1981, 1988; Stool’ et al., 1982; Mulder ct al.. 1984, 1989). In short, neocortical slices were incubated for IS min in 2.5 ml Krcbs-Ringer bicarbonate medium containing 0.1 PM [.‘H]NA to label noradrcncrpic net’ve

terminals selectively. Striatal slices were incubated in medium containing 0.1 PM [‘HIDA and 1 PM [“‘Clcholine. resulting in selective labelling of dopamincrgic and cholincrgic ncrvc terminals, respectively. 2.2. Supc~rfkiatt of bruh slices md addition of drugs

After the slices had been labelled, individual slices wcrc transferred to one of 24 chambers (volume 0.2 ml; 3-4 mg of tissue per chamber) of a superfusion apparatus and subsequently superfused (0.25 ml/min) with medium (gassed with 95% O,, 5% COz) at 37°C. After 40 min of superfusion (i.e. t = 40 min) the superfusate was collected in IO-min samples. At t = 50 min the slices were exposed to electrical stimulation (bipha!;ic square wave pulses, I pps, 2 ms duration) for 10 min to induce calcium-dependent release of the radiolabclled neurotransmitters. For [“H]NA release from ncocortical slices a current of 15 mA was used, and for [‘HIDA and [lJC]ACh release from striatal slices 30 mA. Opioid receptor agonists were added to the superfusion medium 10 min before electrical stimulation of the slices and remained present until the end of the experiment. The cyclic somatostatin analogues to be tested as antagonists were added to the medium 20 min before stimulation. In each experiT!cnt quadruplicatc observations were made. At the end of the expe:imcnt the tissue radioactivity remaining was extracted with 0.1 M HCI. The apparent affinities of the antagonists (PA, values) for p-opioid receptors were determined in experiments in which we used a cumulative dose-response tcchniquc dcvelopcd previously for studying the pharmacological characteristics of presynaptic receptors in supcrfuscd brain slices (Frankhuyzcn and Mulder, !982). In brief, cortical slices labelled with [3H]NA were exposed continuously to electrical field stimulation (1 pps, 2 ms pulses, 15 mA) 40 min after the start: of supcrfusion (t = 40 min). Increasing concentrations, of the Cc-opioid agonist DACO were added cumulatively to the medium at IO-min intervals, from t = 50 to t = 80 min, cithcr in the absence or presence (from t = 20) of one of the somatostatin analogues. In each experiment the inhibitory effect of DAGO was determined (in duplicate) by comparing the electrically evoked release of [“H]NA in the presence of DAGO with the release found in control superfusion chambers not exposed to DAGO.

The radioactivity tissue extracts was counting. The cfllux collection period is

in the supcrfusion samples and determined by liquid scintillation of radioactivity during each IO-tnin cxprcsscd as a pcrccni:~gc of the

3

amount of radioactivity present in the tissue at the start of the respective collection period. To calculate the electrically evoked release of radiolabelled neurotransmitter, the spontaneous efflux of radioactivity was subtracted from the total overflow of radioactivity during stimulation and the 10 min thereafter. The spontaneous efflux of tritium from cortical slices labelled with [“HINA was 0.15-0.20% min-‘; the spontaneous efflux of radioactivity from striatal slices labelled with [“HIDA and [‘4Clcholine was 0.25-0.30% min-’ (“HI and 0.20-0.25% min-’ (‘4C), respectively, of total tissue radioactivity. The electrically evoked release of radioactivity (in excess of spontaneous efflux) from cortical slices ([“HINA) amounted to 3.5-4.5% (of toial tissue radioactivity present at the start of stimulation) and from striatal slices 2.5-4% ([“HIDA) and 6-8% ([ “C]ACh), respectively. A detailed description of the calculation of data obtained in the experiments in which the cumulative dose-response technique was used has been published elsewhere (Frankhuyzen and Mulder, 1982). The concentration-effect curves and pD, values (pD2 = -log EC,,,) were derived from the release data by using the non-linear curve-fitting program ALLFIT. Statistical analysis of the data was done with a two-way analysis of variance, followed by Duncan’s multiple range test, using SPSS/PC + V2.0 (SPSS. Inc). 2.4. Radiochemicals and drugs

I-[7-“HlNoradrenaline (37 Ci/mmol), [7,8-“Hldopamine (47 Ci/mmol) and [methyl- “Clcholine (50 mCi/mmol) were purchased from the Radiochemical Centre (Amersham). Tyr-D-Ala-Gly-(NMeJPhe-Gly-ol (DAGO) was purchased form Bachem. [D-Ser’(O-tbutyl),Leu’]enkephalyl-Thr” (DSTBULET) was kindly

donated by Prof. B.P. Roques (Paris) and 5a, 7a, 8P-(-)-N-methyl-N-[7-(l-pyrrolidinyl)-1-oxaspiro (4,5)dec-8-yllphenylbenzeneacetamide W69,593) by Dr. P.F. Von Voigtlander (Upjohn, Kalamazoo, Mich., USA). CTOP, CTAP, TCTOP and TCTAP were prepared as described preciously (Pelton et al., 1986; Kazmierski et al., 1988).

3. Results At a concentration of 0.1 FM, the p-opioid agonist DAGO inhibited the electrically evoked release of [“H]NA from cortical slices by 55-65%, and this inhibitory effect was reduced by all of the somatostatin analogues tested, in a concentration-dependent manner (fig. 2). By themselves, neither CTOP and CTAP (tested at a concentration of 1 PM) nor TCTOP and TCTAP (tested at 0.1 PM) significantly affected electrically evoked [‘H]NA release or the spontaneous efflux of radioactivity. The antagonist activity of the somatostatin analogues was further analysed with the cumulative doseresponse technique of Frankhuyzen and Mulder (1982). Figure 3 shows concentration-response curves of the release-inhibiting effect of DAGO (0.01-3.0 PM) either in the absence or presence of CTOP (0.1 PM), CTAP (0.1 PM), TCTOP (0.01 PM) or TCTAP (0.01 PM). In the experiments with CTOP and CTAP. pD, values of 7.44 (DAGO alone), 6.48 (DAGO + CTOP) and 6.03 (DAGO + CTAP) were computed, respectively. For the experiments with TCTOP and TCTAP. these values were 7.17 (DAGO alone), 6.31 (DAGO + TCTOP) and 6.30 (DAGO + TCTAP). From these computed pD2 values the following pA2 values were derived for the somatostatin analogues, reflecting their

L

0.1

DA00

DAGO

-

lCTAP

DAGO

0.01

0.1

- +TCTOP

DAGO

’

+ TCTAP

, ol’ thr inhibitory cffcct of the sclcclivc p-opioid agonist DAGO (0.1 pM) on electrically evoked Fig. 2. Anl;lgcrnism by scm~;Uoslatinimidq ‘JOmin bcforc and DAGO IO min hrfore electrical I’IIINA rclcahc from cor1icd slicch. The peptidcs wcrc ;ddcd 10 Ihe supcrfusion mcdiunr _ hlimulrltion. D;lra arc mcuns f S.E.M. of IO- I?. de~crmidons (3 srpuratc cxprriments).

=20

L-3

ODlrp

t

A -xv

ADI~-~T.P !.._-____” -9

-8

-7 109

* -6

.-5

t__

A

D.co.Ic-B

*

D.Go.lCIIP

...I

“’

..-

-9

-E

.,.““’

-7

.‘-‘j

-6

-

-5

tog [DAGO]

[DAG~~

Fig. 3. Concentration-response curves for the inhibitory effect of the p-opioid agonist DAGO (1 nM-3 PM) on electrically evoked [‘H]NA release from cortical slices in the absence or presence of CTOP (0.1 phi), CTAP (0.1 PM), TCTOP (0.01 PM) or TCTAP (0.01 PM). These ‘ltive dose-response technique (see Methods) and the curves were constructed by use of the experiments were carried out using a cumu,.. non-linear curve-fitting program ALLFIT. Data points are means& S.E.M. of 6-8 determinations (3-4 separate experiments).

affinities toward the p-opioid receptors involved: CTOP 7.9, CTAP 8.0, TCTOP 8.7 and TCTAP 8.8. apparent

TABLE

I

Inhibitory effect of the K-opioid agonist U69593 (0.02 PM) on the electrically evoked release of [‘HIDA from striatal slices in the absence or presence of the various somatostatin analogues examined. Data are means+S.EM. from 12-16 determinations (3-4 separate experiments).

[ “HIDA release (as 8 of control)

Peptide

in the presence of

CTOP (1 FM) CTAP (1 PM) TCTOP (0.1 FM) TCTAP (0.1 PM)

TABLE

U69593

Peptide

U69593 + peptide

60.3 f 57.5 * 54.6 5 59.1 f

92.4k3.8 95.7 f 2.5 102.0 f 3.4 103.2 f 3.8

57.2rt3.7 62.Ok3.1 51.2k4.5 57.9 f 4.1

3.0 2.4 2.9 2.7

2

Inhibitory effect on the d-opioid agonist DSTBULET (0.1 FM) on the electrically evoked release of [%Z]ACh from striatal slices in the absence or presence of the somatostatin analogues examined. Data are means f S.E.M. from 12- 16 determinations (3-4 separate experiments). Peptide

CTOP (1 FM) CTAP (1 PM) TCTOP (0.1 PM) TCTAP (0.1 PM)

[“C]ACh release (as 9 of control) in the presence of DSTBULET

Peptide

DSTBULET + peptide

55.7k3.4 62.1 +3.2 53.3f 1.8 54.4k3.2

89.5 f 3.3 92.5 rt 3.5 100.7* 3.8 100.2 f 4.3

56.4k3.1 59.8k3.4 58.5 * 2.5 54.2 rt 2.2

At a concentration of 0.02 PM, the K-opioid agonist U-69593 inhibited the electrically evoked release of [“HIDA from striatal slices by 40-45%. Table 1 shows that this inhibitory effect was not altered by CTOP and CTAP (at concentrations of 1 PM) or by TCTOP and TCTAP (at 0.1 ELM).Similarly, as shown in table 2, the somatostatin analogues did not alter the inhibitory effect of the b-opioid agonist DSTBULET (0.1 PM) on the electrically evoked release of [14C]ACh from striatal slices (38-46% inhibition). At the concentrations tested, the peptides by themselves did not significantly affect the electrically evoked release of [“HIDA and [ “C]ACh or the spontaneous efflux of radioactivity.

4. Discussion In this study the opioid receptor antagonist properties of a number of cyclic somatostatin analogues were assessed using selective functional paradigms in vitro of CL-,S- Lnd K-OpiOid receptor activation in the rat brain. It has been well established that the inhibitory effects of opioids on the depolarization-induced induced release of [‘HIDA and [ 14C]ACh from rat striatal slices are mediated exclusively by K- and S-receptors, respectively, whereas [3H]NA release from rat cortical slices is inhibited by opioids only if they display p-agonist activity (Mulder et al., 1984, 1989, 1991; Schoffelmeer et al., 1988; Werling et al., 1987, 1988). All of the somatostatin analogues tested potently antagonized the inhibitory effect of the selective /.Lagonist DAGO on cortical [3H]NA release in a competitive fashion. None of the peptides by themselves appeared in affect release, in contrast to CTP, which

was shown previously to cause a substantial inhibition of cortical [“H]NA release, apparently via a non-opioid receptor-mediated process (Mulder et al., 1988). It is interesting to note that the Lys residue in position 5 of the CTP molecule appears to be essential for its NA release-inhibiting activity, since in the analogues lacking activity the Lys residue was replaced by either On-r or Arg. Taken together with previous data on CTP (Mulder et al., 19881, it appears that TCTOP and TCTAP (PA, 8.7-8.8) are nearly 10 times more potent as p-antagonists at the functional p-receptors studied here than CTP, CTOP and CTAP (PA, 7.7-8.01, which roughly corresponds with the findings of a recent study (Kramer et al., 19S9) in which these somatostatin analogues were examined as p-antagonists in the guineapig ileum (GPI) preparation. It should be noted, however, that in the latter study CTOP was found to be by far the least potent antagonist, i.e. 10 times less active than CTAP, in contrast to our present finding that these peptides were about equipotent antagonists at the p-receptors mediating inhibition of NA release in brain tissue. Based on the results of the present study it is difficult to give a precise indication of the degree of selectivity of the somatostatin analogues for CL-vs. Sand K-opioid receptors because we did not examine concentrations higher than 1 FM. However, at the concentrations that caused full antagonism of the inhibitory effect of 0.1 PM DA60 on [“HJNA release, i.e. approximately 1 FM CTOP or CTAP and 0.1 FM TCTOP or TCTAP, the peptides did not affect the inhibitory effect of 0.02 p M U69593 on striatal [ ‘HIDA release nor that of 0.1 FM DSTBULET on striatal [‘4C]ACh release. The concentrations of these drugs that selectively activate K- or &rCCCptOrS, t%SpCCtiVCjy, were chosen to give a sub-maximal inhibitory effect (see De Vries et al., 1989; Mulder et al., 1991). Therefore, assuming that the somatostatin analogues are indeed able to act in the micromolar concentration range as antagonists at A- and/or K-reCCptOrS, one might make a conservative estimate that these compounds are at least lOO- to 200-fold selective for ELopioid receptors. However, other studies with ligandbinding assays in brain membranes or the GPI preparation (Pelton et al., 1986; Shook et al., 1987; Kazmierski et al., 1988; Kramer et al., 1989) indicate that the degree of selectivity is likely to be far greater. In conclusion, the cyclic octapeptide somatostatin analogues CTOP, CTAP, TCTOP and TCTAP all are potent antagonists at the CL-opioid receptors mediating presynaptic inhibition of NA release in rat brain but not at the K-receptors and &receptors mediating inhibition of striatal DA and ACh release, respectively. The most potent of these analogues, TCTOP and TCTAP, appear to have an affinity for p.-opioid receptors similar to that of naloxone fpAz about 8.6; Mulder et

al., 19911, but have a much greater selectivity than the latter.

This work was supported by US. Public Health Service Grant NS 19972. The authors also thank Mr. Henk Nordsiek for preparing the figures.

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h

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