Neuropeptides 6: 321-329, 1985

INTERACTION OF DYNORPHIN WITH KAPPA OPIOID RECEPTORS IN BOVINE ADRENAL MEDULLA

Michel Dumont and Simon Lemaire Departement de Pharmacologic, Centre Hospitalier Universitaire, Sherbrooke, Quebec, Canada JlH 5N4 (reprint requests to S.L.)

ABSTRACT

Dynorphin (Dyn) and various prototypic K opioid ligands were tested for their ability to bind to opioid receptors in a membrane preparation of bovine adrenal medulla and to modulate the release of catecholamines (CA)

om isolated adrenal chroma fin cells. F’H]- thylketocyclazocine ’

Saturation binding studies with e ,(I HI-E$+C) were5p erformed at 37’C for 30 min

in the2pres nce of [D-Ala ,Me-Phe , %

Gly-01 I-enkephalin (DAGO) and [D-Ser , Thr ]-Leu-enkephalin (DSLET), two specific ligands for cross- reacting u and 6 opioid receptors, respectively. Scatchard plot analysis of the data revealed the presence of two receptor sites: a high affinity binding site (K) with a KD of 0.66 nM and a Bmax of 12 pmoles/g protein and a low affinity binding site ( k2) with a KD of 11 .l nM and a Bmax of 56 pmoles/g protein. The presence of K opioid receptors in the membrane preparation was also supported by competition studies. selective K opioid ligands,

U-50,4888 and gyn-(l-13)) two were potent inhibitors of [ HI-EKC binding

with Ki (high affinity binding sites) of 2.5 and 2.3 nM, respectively. Among the various ligands tested for each class of opioid receptors (p, 6, K), U-50 ,488H and Dyn-(l-13) were the most potent inhibitors of the acetylcholine-evoked CA secretions from isolated adrenal chromaffin cells with XC U-50,4821

of 0.31 and 1.14 uM, respectively. The inhibitory effect of was significantly antagonized by diprenorphine and MR-2266, two

opioid antagonists with a high affinity for the k opioid receptor. Among various fragments of Dyn, Dyn-(l-8) and larger N-terminal peptides retained a sign&ficaqt part of the potency of Dyn-(l-13) (> 6%) while Dyn-(l-51,

[Arg , Phe I-Met-enkephalin and Dyn-(6-13) were relatively ineffective (relative potency < 1%). These results suggest a putative modulatory role for Dyn in the adrenal medulla, possibly mediated through the stimulation of

K opioid receptors.

INTRODUCTION

The adrenal medulla is an important source of endogenous opioid peptides (1). Among these peptides, Dyn (or Dyn A; Ref. 2), a specific endogenous opioid ligand (21, was shown to be localized in an adrenal cell population enriched in noradrenaline whereas Leu-enkephalin (Enk) was rather confined

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into a cell population enriched in adrenaline (1). In order to better define the role of these peptides, we have recently characterized the adrenal opioid receptors (4) and demonstrated an heterogeneity of opioid receptor types. Herein, the use of prototypic agonists and antagonists with high affinity for the K opioid receptor has allowed us to make a close correlation between the binding characteristics of this receptor type and the modulation of CA secretion from isolated adrenal chromaffin cells.

MATERIALS AND METHODS

The binding assay was performed with bovine adrenal3 membrane preparations as described earlier (4). The binding of [ HI-EKC was conducted.at 37’C for 30 min with 2 ml suspensions of the membrane preparation (0.5 mg protein/ml in 50 mM Tris-HCl buffer, pH 7.4) in presence or absence of the indicated opioid ligands. The bound tritiated ligand was separated fro9 free by filtration through GF/B Whatman filters. Saturation curves with [ HI-EKC were carried out in the presence of 0.1 pM DAGO and 0.1 uM DSLET in order to block cross-reacting u and 6 opioid receptors, respectively (5). The secretion assays were also performed as described earlier (6). Bovine adrenal chromaffin cells were isolated by successive digestions of the adrenomedullary tissue with collagenase. They were kept at room temperature over night under 02/C02 (95%/5%) and incubated the next day in the presence opioid compounds.

or absence of acetylcholine (50 gM) and the indicated Secretion was carried out at 37 C for 5 min in Krebs

buffer, pH 7.4 and CA werf monitored in the incubation medium by spectrofluorometry (7). [ H I-EKC (16.4 Ci/mmol) was purchased from New England Nuclear, Boston, MA. U-50,488H (trans - (+I - 3,4-dichloro- N-methyl-M [ 2 -( 1-pyrrolidinyl) cyclohexyl 1 benzeneacetamide methanesulfonate hydrate) was obtained from Upjohn Company (Kalamazoo, MI). Mr-2034 ((-)-(IR,5R,9R,2”S)-5,9-dimethyl-2 ‘-hydroxy-2_tetrahydrofurfuryl- 6,7-benzomorphan D-tartrate) and Mr-2266 ((-)-(IR,5R,9R>-5,9-diethyl- 2-(3-furylmethyl) 2’- Dr. H.

hydroxy-6,7-benzomorphan) pere gene&ously provjded by Merz (Boehfingeg Ingelheim). [D-Ala ,Me-Phe , Gly-01 ]-Enk

(DAGO) and [D-Ser , Thr ]-Leu-Enk (DSLET) wele obtqined from Peninsula Labor2tories5(San Carlos, CA). [Arg ,Phe ]-Met-Enk and [D-Ala D-Leu ]-Enk were purchased from Bachem (Torrance, CA). B-endorpdin, Dyn-(l-13) and Dyn fragments were synthesized in our laboratory as described by Turcotte et al. (8). All binding and secretory experiments were performed three times induplicates and the results are expressed as the means + SEM.

RESULTS

3 Fig. 1 illustrates the binding of increasing concentrations of

[ H]-EKC in the presence of specific blockers for cross-reacting p and 6 opioid receptors. High affinity opioid binding sites for the tritiated ligand were detected with a KD of 0.66 nM and a Bmax of 12 pmoles/g protein. Scatchard plot analysis of the data (Fig 1, inset) also demonstrated the presence of a low affinity binding site with a KD of 11.1 nM and a Bmax of 56 pmolesfg protein. The high and low affinity binding sites were referred as the K and (4).

K2 opioid receptors, respectively

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40 -

0 40 00 pmol bound/g protein

0 3 6 9 12 15 1s

[3~1-~~~~~~~~~~~~~~~~~~~~ Lml

Fig. 1 Dose response curve of the binding of 13H]-EKC to bovine adrenal membranes. and Methods”

Binding experiments were performed as described under “Materi3ls and the non-specific binding (in the presence of 10 M

levallorphan) was subtracted for each concentration of the tritiated ligand. Each point is the mean 2 SEM of three duplicated experiments. TJe inset represents the Scatchard plot of the data. B/F: bound [ HI-EKC (pmoles/g)/free ligand (nmoles).

Table 1. binding of

In$ibitory potencies of U-50,488H and opioid peptides on the [ HI-ethylketocyclazocine (2 nM) in a membrane preparation of

bovine adrenal medulla.

Canpeting Ki (high aff.) Ki (low aff.) Ki (high aff.)/ ligand (nM) ( nM) Ki (low aff.)

u-so, 488~ Dyn-( l-13) &En$orphjn [Arg -Phe I-Met-Enk

2.5+0.1 765 + 97 306 2.350.2 269 z 57 116 7.1T1.9

>5,000

Various opioid compounds were also tested for their ability to displace t3H]-EKG binding (Table I). Detailed dose-response competition curves with U-50 ,488H and Dyn-(l-131, two selective K opioid agonists (3,9), were biphasic, with Ki high affinity of 2.5 nM and 2.3 nM and Ki low affinity of 765 nM and 269 nM, respectively. The high affinity binding site represents 8-10% of total binding activity and may be considered as the K component. The high ratio between the Ki (low affinity) and Ki (high affinity) of U-50 ,488H confirms the selectivity of this compound for K opioid receptors. B-endorphin was also a potent inhibitor of [ HI-EKC binding with Ki of 7.1 nM. However, no distinction could be made between the compound to the high fnd low affinity binding sites. was a poor inhibitor of [ HI-EKC binding (Ki: > 5,000).

Various opioid compounds prototypic to distinct classes of opioid receptors (p, 6 , K) or belonging to distinct families of endogenous opioid peptides (B-End, Dyn, Enk) were tested for their ability to modulate the release of CA from isolated bovine adrenal chromaffin cells. compounds was able to modulate the basal secretion

None of thesg (0.4 + 0.1 nmole/lO

cells). The presence of acetylcholine (50 pM) induced a lo-fold stimulatiog of CA secretion over the basal value (from 0.4 to 4.2 nmoles per 10 cells). However, when the stimulation with acetylcholine was conducted in the presence of increasing concentrations of opioids agonists, a dose-dependent inhibition of CA secretion was observed. Among the various opioid alkaloids tested, the prototypic K agonists, U-50,488H, MR-2034 and EKC, were the most potent inhibitors of acetylcholine-induced CA secretions with IC5U of 0.31, 1 .93 and 7.2 pM, respectively (Fig. 2A; Table 2). The prototyprc p and 6itgands, dihydromorphinone (DHMone) and DSLET, were poor inhibitors (fC50 > 10 Mf.

Dose-response inhibitions of the acetylcholine-evoked release of CA were also obtained with the endogenous opioid peptides and their relative potency is indicated in Table 2. Dyn-(l-13) was the most potent inhibitor with an IC50 of 1.14 pM (Fig. 2, Table 2). l3-Endorphin and n-neoendorphin had ;A;;#i;;ef;nge potencyMJ:_CJU of 50 and 69 uM, respectively) whi.le

nk and Leu-Enk were relatively ineffective

(IC50 > 10

_z Met-Enk, M). Various fragments of Dyn were also tested (Table 2).

The minimal core for inhibition of CA secretion resides inside the N-terminal fragment (Dyn-(l-8) with a rglat$ve potency of 6% as compared with Dyn-(l-13)). Dyn-(l-51, [Arg ,Phe ]-Met-Enk and Dyn-(b-13) had less than 1% of the potency of Dyn-(l-13) itself. On the other hand, the addition of one or three residues to Dyn-(l-8) to give Dyn-(l-9) or Dyn-(l-11) greatly increased the potency of the peptide (from Table 2). Finally, Dyn-(2-13) (or Des

P% to 25% and 75X, respectively; Tyr -Dyn-(l-13)) retained 9% of the

activity of Dyn-(l-13).

The specificity of the action of U-50,488H was verified by the ability of various opioig antagonists to block its effect on CA secretion (Fig. 2B). Nal trexone ( 10 M) did not significantly antaqnize the inhibitory ef_fpct of U-50,488H while diprenorphine (10 M) and MR-2266 (10 M) significantly blocked the activity of U-50,488H. Higher concentrations of these three opiate antagonists produced their own inhibitory effects on CA secretion (data not shown). Interestingly, MR-2266 was reported to have a high affinity and some selectivity for the k opioid receptor (10) while

324

lo -9 ’ ’ a 10 -6 lo-! 10 -9 10 -5 l0

-4

OPIOID AGONIST [Ml

a. U-50.466H

-Dyn-(l-13)

,MR-2034

0 O-End

,DSLET

b DHMOne

60

60

*Control

oNaltrexone [lO-sMl

,Dlprenorphlne [~o~‘MI DMR-2266 [+O-‘Ml

-u ,

1o-g lo-* lo-’ 1o-6 i-5 A.4

U-50.466H [bfl

Fig. 2. (A) Effects of increasing concentrations of various opioid agonists on the acetylcholine-evoked release of catecholamines from isolated bovine adrenal chromaf f in cells (B-End : B-endorphin; DHMone: dihydromorphinone). (B) Reversal of the inhibitory effect of U-50,488H in the absence or presence of opiate antagonists.

diprenorphine showed a high affinity but no selectivity for this same receptor (11).

DISCUSSION

The presence in the adrenal medulla of high affinity stereospecific opiate binding sites was first reported by Chavkin et al. (12). Thereafter, various studies have indicated that these sites were either different from all other known opioid receptors (p, 6, K, Ref. 13) or that they were a mixture of all opioid receptor types (14). We have recently made a full characterization of the opioid binding sites in bovine adrenal medulla and found the presence of various types of opioid receptors: two with high affinity and low density (K, 6 ), one with high affinity and high density (u) and two with low affinity and high density (K~,u). Herein, the physiological significance of the K receptor was further examined.

325

Table 2. Relative inhibitory potency of opiate agonists on the acetylcholine (50 pM)-evoked release of CA from isolated adrenal chromaffin cells.

Opiate compound Ref;;;or :(&’ Ref. potencyb (X1

Prototypic agonists U-50,488H MR-2034 EKC Dihydrjmorpbinone [D-Ser ,Thr ]Leu-Enk Opioid peptides Dvn-( l-13 > SIendorphin c1 -n oend rphin f Arg’, Phe’ IMet-Enk Met-Enk Leu-Enk Dyn-( l-8) Dyn-( l-9) Dyn-( l-11) Dyn-(2-13) Dyn-(6-13)

(K) 0.31 + 0.06 100 it; 1.93 7 0.26 16

7.20 5 1.60 4 ( 1J) >I00 <0.3 (6) >lOO <0.3

1.14 + 0.33 100 50 T 9 2 69 7 25 2 >lOO <1 >lOO <l >lOO <l

18.4 + 2.9 6 4.5 T 1.5 25 1.5 + 0.2 75

12.3 : 2.8 9 > 100 <l

a The ZC50 represents the concentration which gives half-maximal Eesponse.

The potency is relative to that of U-50,488H (first section) or Dyn-(l-13) (second section).

Previous studies have already indicated that B-endorphin, Dyn, levorphanol and other opioid compounds were able to inhibit the acetylcholine-evoked release of CA from isolated adrenal chromaffin cells (6, 15-17). The release of CA in response to high potassium concentrations or to veratridine was not affected by the opioid compounds (6, 15). Among the various opioid compounds tested, Dyn was the most potent inhibitor of acetylcholine-induced CA secretion; but, since the inhibition was obtained for most compounds at pmolar leve 1s and since it was not antagonized by na loxone, it was concluded that this effect was more likely a non opiate effect (6, 17). Thereafter, Dyn was isolated from bovine adrenal medulla (1) and recognized to be a selective ligand for the K opioid receptor (3). Herein, the possibility that the naloxone-insensitive effect of Dyn in the adrenal medulla be mediated by the stimulation of specific K receptors was raised. Our data indicate t3at the most selective K opioid ligands can both displace the binding of [ HI-EKC from its high affinity binding site ( K receptor) and inhibit the acetylcholine-induced CA secretion from isolated chromaffin cells. Therefore, U-50 ,488H, the most selective

K agv ist (9)) was very potent in displacing the high affinity binding site for [ HI-EKG (Ki: 2.5 nM, Table I) and inhibiting the acetylcholine-evoked CA secretion (IC50: 0.31 PM; Fig. 1A). In addition, all the specific K opioid agonists tested, including U-S0,488H, Dyn-(l-133, MR-2034 and EKC,

326

were also potent modulators of CA secretion, whereas the prototypic ligands to p and 6 receptors were essentially inactive.

In order to determine the structural requirements of Dyn for inhibition of acetylcholine-induced CA secretion, various Dyn related fragments were tested and their activity was compared with that of Dyn-(l-13) (Table 2). The inhibition of acetylcholine-e,voked CA secretion by these peptides was structure-related. The minimum core for inhibition of CA secretion was conta’ned

& ythin the N-terminal fragment Dyn-(l-8) (Table 2). Leu-Enk and

[Arg ,Phe ]-Met-Enk were less than 1% as potent as Dyn-(l-13). Conversely, the C-terminal portion of Dyn that is Dyn-(6-13) had relatively no significant activity. If we compare the activity of these peptides in the present system with those reported in the guinea pig ileum preparation (18)) we can see that the major requirements for the particular potency of Dyn-related peptides are the same. In fact, it was noted that Dyn-(l-8) is the minimal core for the selective stimulation of Dyn receptors in the guinea pig ileum. Fur thermore, large increases in the activity were observed when Dyn-(l-9) and Dyn-(l-11) were compared with Dyn-(l-8). This parallelism obtained between the activity on the classical guinea pig ileum preparation and the isolated chromaffin cells indicates that the inhibition of CA secretion by Dyn and related peptides in our assay may be mediated by the stimulation of some specific opioid receptors.

The relative high potency of Dyn-(2-13) (9% as compared with Dyn-(l-13)) in inhibiting the acetylcholine-evoked CA secretion can be compared with the high potency of this peptide in other systems. Dyn-(2-13) had a high potency in inhibiting the analgesic effects of morphine (191, producing motor function activity in the central nervous system (20) and increasing feeding in rats (21). The specific activation of the adrenal K opioid receptors by Dyn may not necessitate the presence of the Tyr residue. Eventhough Dyn-( l-13) was the most potent peptide tested, the relative high concentration of this peptide to produce half-maximal inhibition of acetylcholine-evoked CA secretion may be explained in many different ways. Firstly, our preparation of isolated adrenal chromaffin cells may contain high peptidase activity. Such enzyme activity would greatly decrease the effective concentration of Dyn. Secondly , with a cell preparation obtained by collagenase digestion, there is always a possibility of some receptor modifications; however, due to its high responsiveness to various secretagogues, our preparation is considered as highly functional (6). Finally, it is quite possible that in “in viva” situation, the concentration of Dyn reaches high levels at specific receptor sites located closely to the site of Dyn secretion.

One of the most important criteria to attest the specificity of a biological effect is its blockade with selective antagonists. The present study shows that naltrexone, a u selective opioid antagonist (221, does not significantly inhibit the activity of U-50,488H on CA secretion while diprenorphine and MR-2266, two opioid antagonists with high affinity for the K receptor, are potent blockers. The possible involvement of the high affinity (instead of the low affinity) K opio id binding site in the modulation of CA secretion is supported by the particujar high potency of U-50,488H and Dyn-(l-13) to diplace the binding of 6 HI-ETC to its high affinity receptor site (Table 1). Furthermore, [Arg , Phe ]-Met-Enk and B-endorphin were reported to be selective ligands of the K 2 binding site

327

(23,24); in the present study, these two latter compounds have little modulatory activity on the release of CA (Table 2). In conclusion, the opioid inhibition of acetylcholine-induced CA secretion from isolated bovine adrenal chromaffin cells is more likely a selective k opioid activity, not involving the n opioid binding site.

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