Neuroscience Vol. 7, No. 4, pp. 1007 to 1013, 1982 Printed in Great Britain.

030~4522/82/o41~7-07a3.oo/o Pergamon Press Lid

Q 1982 IBRO

SELECTIVE INHIBITION OF ‘MOTOR ENDPLATE-SPECIFIC’ ACETYLCHOLINESTERASE BY

fi-ENDORPHIN AND RELATED PEPTIDES

L. W. HAYNES and M. E. SMITH

Department of Physiology, The Medical School, Vincent Drive, Birmingham B15 2TJ, U.K.

Abstract-The effects of /I-endorphin (lipotropin 61-91) and related naturally-occurring peptides upon acetylcholinesterase. activity in rat hind-limb muscles was investigated. l-endorphin weakly inhibited the activity in a plasma membrane-enriched fraction. The inhibition by /I-endorphin of the membrane- associated acetylcholinesterase was less marked when the fractions were prepared from muscles which had been denervated 4-6 days previously. The membrane-associated acetylcholinesterase was solubilised from normal muscle preparations and separated by sucrose density gradient centrifugation into three major peaks (16S, 10s and 4.9. /&Endorphin inhibited the activity in the 16s peak but not that in the 10s and 4S peaks, whilst tensilon, a competitive inhibitor of acetylcholinesterase, inhibited the activity of all three peaks. /?-Endorphin inhibited the 16s activity in a concentration-dependent manner and its action was partly prevented if naloxone was added simultaneously. Purified natural porcine and bovine /?-endorphin were equipotent in terms of effective concentration range but the maximum inhibition was greater with the bovine peptide. /?-Lipotropin was approximately 4 times less potent than b-endorphin, whilst (Y-fragment (lipotropin 61-87) was 100 times less potent. Prolonged treatment with collagenase did not reduce the catalytic activity of 16s acetylcholinesterase, but it was no longer susceptible to the inhibitory action of b-endorphin. Kinetic studies indicated a complex type of inhibition by /I-endorphin (hyperbolic Lineweaver-Burke plot). Methionine enkephalin inhibited acetylcholinesterase in a weakly non-competitive nianner and its action was not abolished if the enzyme was predigested with collagenase.

/?-Endorphin produces a novel form of inhibition of acetylcholinesterase, acting only on the 16s (Al2 or ‘motor endplate-specific’) form of the enzyme. The findings are discussed in the light of evidence that /3-endorphin-related immunoreactivity is expressed in motor nerve axons in the immature rat.

Lipotropin C-terminal immunoreactivity, represent- ing predominantly lipotropin 61-91 (fl-endorphin), is confined in the adult rat central nervous system to nerve cells in the hypotha1amus3’ and in a number of sites to which these cells project in the forebrain and brainstem.2*27*28 Recently, however, neurones within cultures of embryonic rat spinal cord tissue have been shown to contain immunoreactivity to antisera di- rected against fl-endorphin’ ’ and fl-lipotropin.’ 5 These observations indicate that immature spinal neurones may have the capacity to synthesise endor- phins and their precursors although these peptides are absent from this region in adult life. Spinal immuno- reactivity to p-endorphin-related peptides in the im- mature rat is present amongst other sites in ventral horn neurones and in motor nerve axonsi but their physiological function therein is unknown. We have therefore begun to investigate whether any of these peptides influence the functional properties of skeletal muscle. Previous work9 has shown that /I-endorphin potentiates the response of denervated skeletal muscles to acetylcholine. Recently, we have demon- strated that p-endorphin is a specific inhibitor of the

Abbreviation: AChE: Acetylcholinesterase; EDTA, ethy- lenediamine tetra-acetate.

16s form of acetylcholinesterase (acetylcholine hydro- lase, E.C. 3.1.1.7, AChE).” The 16s oligomeric form of the enzyme occurs exclusively in the motor end- plate regions of adult rat skeletal muscle.’ In this report, we describe the nature of this inhibition and compare the potency of physiologically active natur- ally-occurring peptides related to /I-endorphin in their inhibitory action on the enzyme.

EXPERIMENTAL PROCEDURES

Animals

Female Sprague-Dawley rats weighing approximately 150 g were used.

Surgical procedure

Denervation of lower hind-limb muscles was carried out under ether anaesthesia by unilateral section of the sciatic nerve, high in the thigh, 4-6 days prior to the experiment. A 2mm section of nerve was removed in order to delay reinnervation. Muscles of the lower limb on the operated side showed signs of atrophy. The contralateral muscles were used as controls.

Preparation of muscle fractions

The animals were killed by a blow on the head and the lower hind-limb musculature was removed and immedi-

1007

1008 L . W . Haynes and M. E. Smith

ILl ,52-

Z ADH m

¢, , o

? i i

' I " i i I ,

16 ' -zb 3b" z,,b " 5b Fnno.

5 -

.c 4-

-6 ~3-

laJ xz 2 - t_)

165 105 45

4-

~3-

c v

Z m

10 20 30 40

P

56 Ft. no.

Fig. 1. Effect of inhibitors on the sedimentation profile for solubilised hind-limb muscle acetylcholinesterase activity. a. Typical profile (solid line) in which AChE specific ac- tivity (nmol substrate hydrolysed/min/mg protein) is plot- ted against fraction number. Enzyme activity was calcu- lated from the total AChE activity of the extracted hom- ogenate by integration of the curve. Position of meniscus is marked, m. Dotted lines show sedimentation positions of marker enzymes: fl-galactosidase (Z) and alcohol dehydro- genase (ADH). Their activities are plotted on an arbitrary scale of optical density with zero activity at the baseline. Solid bars below the baseline indicate 16S, 10S and 4S peak fractions retained (determined for each gradient sep- arately) for experiments shown in b. b. Effect of porcine fl-endorphin (10 v M) (solid bar) and tensilon (1.5 x 10 -6 M) (hatched bar) on the activity of AChE in fractions containing peak enzyme activity at 16S, 10S and 4S. Open bars show enzyme activity without inhibitor. Error lines show S.E.M. calculated from the results of 4-13 estimations. Inhibition of 16S AChE by /Lendorphin,

ately placed into 0.05 M tris HC1 buffer (pH 7.3) on ice. Connective and fatty tissue was dissected away and the muscles were minced and then homogenised using a Poly- tron P10 homogeniser (90 sec, setting 5) at a concentration of 0.2 mg/ml buffer, in a total volume of approximately 12 ml. The homogenate was centrifuged in an MSE Super- speed 50 centrifuge at 30,000 g for 20 min at 4°C. The pellet was resuspended in 2 ml buffer.

Separation of oligomeric forms of acetylcholinesterase

Homogenates were prepared as above, using a high ionic strength buffer containing detergent, similar to that used by Vigny, Koenig & Rieger. 26 The composit ion of the buffer was 1 M NaC1, 0.05 M Tris HC1, 0.2 m M EDTA and 0.5~o Triton X-100 corrected to pH 7.3 at 4°C. Homogen- ates were left to stand in a freezing mixture for 1 h, and were then centrifuged at 15,000g for 20min at 4°C. The supernatants were retained and the pellets were resus- pended with gentle homogenisat ion (Polytron P10, setting 1) in 1 ml solubilising solution. The solubilisation and cen- trifugation steps were repeated three times, and the super- natants were combined and centrifuged at 30,000g for 30 min at 4°C. 71~o total specific activity was solubilised in this way. The homogenate was used immediately or frozen at - 7 0 ° C for up to 3 months before use. Activity of hom- ogenates did not change after storage, but the activity of 16S AChE declined if the sample was thawed more than once. 13.5 ml (95 m m tube length) gradients of 5 20~o su- crose in solubilising buffer were prepared using a MSE gradient generator and LKB peristaltic pump (flow rate of

0.3 ml/min). 0.2 ml aliquots of homogenate were layered onto the gradients. In some experiments, bovine fl-endor- phin was added to the homogenate prior to separation at a concentration of 10 pmol /mg protein. The gradients were placed in an S.W.40 t i tanium swing-out rotor and centri- fuged in a Beckmann L5-65 centrifuge at 182,850g,, (38,000 r.p.m.)for 18.5 h at 2°C. Gradients were calibrated using enzyme markers: fl-galactosidase (E.C. 3.2.1.2.3, sedi- mentat ion coefficient 16.14S 16 and alcohol dehydrogenase (E.C. 1.1.1.1, sedimentation coefficient 4.8S. 2s The 16S, 10S and 4S peaks were identified and the peak fractions from 4 gradients were pooled (Fig. 1). Inhibitors (peptides or tensi- lon) were incubated with the assay mixture at room tem- perature for 20 min. Incubation of 16S AChE with colla- genase (1000u/ml enzyme solution) was carried out at 37°C for 3 ~ 4 5 rain. For experiments with collagenase, EDTA was omitted from the medium and 30 m M CaCI 2 and 5 0 m M MgCI 2 included. The tryptic activity of the collagenase sample was less than 0.15~o.

Protein and enzyme determinations

Protein was determined by the method of Lowry, Rose- brough, Farr & Randall 19 using bovine serum albumin (Fraction V powder, Sigma) as standard. The activity of 5'-nucleotidase (E.C. 3.1.3.5) was determined by the method of Mitchell & Hawthorne 22 and the activity of AChE by the method of Ellman, Courtney, Andres & Feathers tone3

*P = 0.06. c. Effect of bovine fl-endorphin (10 pmol/mg protein), added to the extract before density gradient cen- trifugation, on the profile of AChE specific activity 5 (open circles) and 30 (squares) hours after fractionation. Filled circles show profile of activity from the same extract in the absence of fl-endorphin. Scales as in a. Sedimentation pos-

ition of fl-galactosidase shown (Z).

Inhibition of 16s acetylcholinesterase by @endorphin 1009

Table 1.

Preparation

Acetylcholinesterase nmol acetyicholine

hydroiysed/ % Fraction min/mg protein n P inhibition

Unoperated Unoperated + iO_’ M

~-endorphin Unoperated Unoperated + 10e9 M

/&endorphin Unoperated f 10m7 M

fi-endorphin Denervated Denervated + lo-’ M

/I-endorphin Denervated Denervated + 10e7 M

fi-endorphin

Supematant 26.42 + 0.16 25.98 + 2.13

Pellet 22.87 f 0.61

19.76 k 0.43

18.40 + 0.42 Su~rnatant 6.20 & 0.35

5.88 k 0.35 Pellet 13.82 + 0.41

11.76 f 0.20

4 6 n.s.

6

4 <0.02 13.56

6 <O.Ol 19.34 6

6 n.s. 10

4 <0.02 9.5

Effects of porcine B-endorphin on the acetylcholinesterase of supernatant and pellet frac- tions prepared from left hind-limb muscles from unoperated rats or from rats after left sciatic nerve transection. /3-endorphin was added to the assay mixture 20min prior to estimation. Denervations were performed 4-6 days prior to the assav. Values are eiven as the means + S.E.M. n is the number of experiments and P is the probability leiel determined using Students ‘r’ test.

Materials

Natural purified porcine and bovine /?-endorphin and C’-fragment of lipotropinz0q24 were generously provided by Dr. D. G. Smyth, ovine ~-li~tropin by Professor C. H. Li and naloxone by Endo Laboratories. E. coii #I-galactosi- dase, horse liver alcohol dehydrogenase and collagenase type III were obtained from Sigma Co., met5-enkephalin from Bioproducts (Brussels) and tensilon (Edrophonium chloride) from Roche.

RESULTS

E@ect of j?-endorphin on soluble and membrane-bound acetylcho~~nesterase in normal and denervated muscle

Following centrifugation of homogenates at 3O,OOOg, 82% of the activity of S-nucleotidase was detected in the pellet, indicating that this fraction con- tained most of the plasma membrane fragments. The specific activity of the AChE in the pellet and super- natant fractions obtained from both normal and denervated muscles is shown in Table 1. The total activity of the denervated muscles was approximately half that of the normal muscles with a greater propor- tion being recovered in the pellet fraction (Table 1).

/LEndorphin inhibited the activity of AChE in the pellet fractions obtained from either normal or dener- vated muscle, although the inhibition was less marked in the case of denervated muscle. The soluble enzyme activity (supernatant) was unchanged when assayed in the presence of /?-endorphin in both normal and denervated muscle (Table 1).

Effect of /3-endorphin and tensilon on the activity of deferent ol~~orn~s of acetylchoiinester~e

The membrane-associated enzyme was solubilised, and fractionated using sucrose-density gradients and

three major peaks of activity were resolved. Figure la

shows a typical profile of activity seen after the separ-

ation procedure. Major peaks of AChE appear at

approx. 16, 10 and 4s. Minor ‘shoulders’ are evident in this and in most profiles at 18.5, 12.5, 8 and 6s. Fractions contained in the peaks were pooled and the AChE activity was assayed in the presence and absence of Bendorphin (10m7 M) or tensilon

(1.5 x 10e6 M). Figure lb shows that ~-endorphin in- hibited the activity of the 16s form of AChE but did not affect the activity of the 10 or 4s forms. Tensilon, on the other hand, inhibited the activity contained in all three peaks.

In some ex~riments, ~-endorphin (10 pmol/mg protein) was added to the muscle homogenate prior to the separation. Figure lc compares the separation profiles of enzyme activity obtained in the presence and absence of @-endorphin. Inhibition of the 165 AChE activity (assayed 5 h after s~imentation) is seen, as when the peptide is added to the appropriate fractions before assaying the enzyme, indicating that the added fi-endorphin copurified with the 16s AChE and may therefore have remained bound to it. When the fractions were assayed 30 h after the separation (fractions kept at 4”C), however, an inhibition of the enzyme activity was barely detectable (Fig. lc).

Efiect of naloxone

The AChE activity of fractions pooled from the 16s peak was determined in the presence of different con- centrations of porcine fl-endorphin. Figure 2a shows that the inhibition of 16s AChE by j?-endorphin was partially prevented by the simultaneous addition of the opiate antago~st naloxone with the peptide. Nal- oxone alone, at the same concentration (lo-’ M) did not influence 16s AChE activity.

L. W. Haynes and M. E. Smith

Pept ide concentmt ion &I)

Fig. 2. Concentration dependence of the effects of /I-endor- phin and met’-enkephalin on the specific activity of 16s acetylcholinesterase preparations before and after incuba- tion with collagenase (1000 p/ml) at 37°C and reduction of inhibition with /I-endorphin by naloxone (lOes M). The results were obtained from preparations made from 5 gradients (4 animals). a. Inhibition of intact 16s AChE by porcine j?-endorphin (0). inhibition by porcine fi-endor- phin with naloxone added simultaneously (m), activity of collagenase treated enzyme in the presence of porcine j-endorphin alone (0). b. Inhibition by met5-enkephalin

before (a) and after (0) collagenase.

Efiect of collagenase digestion

It has been demonstrated that on prolonged treat- ment of the high molecular weight forms of ACHE with purified collagenase at 37°C the tetrameric forms of the enzyme, obtained from Electrophorus electric organ, can be liberated from the collagen tail3 The enzyme retained its full catalytic activity after this treatment. An attempt was made to show whether the selective inhibition of the 16s form of the enzyme by /I-endorphin depends on its attachment to its collagen tail. Therefore the separated 16s enzyme fraction was incubated with collagenase and the effects of &endor- phin and met&enkephalin on the activity after the digestion was studied. Figure 2a shows that the cata- lytic activity of 16s AChE from skeletal muscle is not reduced following collagenase digestion. However, the enzyme was shown to be completely resistant to the inhibitory action of B-endorphin after the collagenase treatment. In contrast, the weak inhibitory action of met-enkephalin was not changed following collage- nase treatment (Fig. 2b).

E&t of /I-endorphin and mets-enkephalin at different substrate concentrations

Figure 3a shows substrate concentration curves for 16s AChE in the presence of concentrations of por- cine b-endorphin or metenkephalin which produced half maximum inhibition of the enzyme., and in the absence of inhibitor. When @ndorphin was present the concentration curve exhibited a sigmoid devi- ation. Lineweaver-Burke plots of the data are shown in Fig. 3b. The plot obtained in the of iahibi- tor was linear and the K, value was O.fdmM. When fl-endorphin was present, however, the plot exhibited a hyperbolic profile indicating a complex, possibly

allosteric, inhibition. The inhibition of 16s AChE by met-enkephalin, on the other hand, was shown to be probably of a simple non-competitive kind.

Effects of different peptides on 16s acetylcholinesterase activity

The AChE activity of a 16s enzyme preparation

was determined in the presence of different concen- trations of /I-endorphin or the immunologically- related peptides /I-lipotropin or C-fragment. Porcine and bovine lipotropin C-terminal peptides were also compared in this respect. Figure *a-c) shows the effects of different concentrations of these peptides on the enzyme activity. Porcine /I-endorphin was potent over a similar concentration range as bovine fl-endor- phin, with a concentration of approximately lo-’ M giving half maximum inhibition. The maximum inhi- bition by bovine b-endorphin, however, was greater

a

V

b

l/V 20r I

Cl

15-

‘O- / I I

0

5 0 L A4

10 20 30

1 /PiI

Fig. 3. Activity of 16s acetydeI@inesterase at different sdb- strate concentrations in thri ParpGnae and a;‘lbawilQ of par- tine &endorphin (2 x lO’“)NI) (0) and m~~~~~ (lOVs M) (A). Enzyme alone (0). a. h&‘ltadhaot~l’&mWr

plots. b. Lineweaver-Burke plots.

Inhibition of 16s acetylcholinesterase by jl-endorphin 1011

a 37 13 -End

b 3.

C

C’-Fragment

Peptide concentration (M)

d log (v/n-v)

-log [Ob-EndI

Fig. 4. Concentration dependence of the effects of endorphins and /?-lipotropin on the specific activity of acetylcholinesterase. Enzyme preparations as for Fig. 2. a. bovine /?-endorphin (e), porcine A-endorphin (m), curve redrawn from Fig. 2a. b. C-fragment bovine (0) and porcine (m) peptides. c. b-lipotropin. d.

Hill plot from data in a. for bovine j-endorphin.

than that seen with porcine /I-endorphin. C-fragment of lipotropin (bovine and porcine peptides) was over lOO-fold less potent than fl-endorphin in producing half maximum inhibition, whilst /I-lipotropin was nearly as potent as /I-endorphin. I.C.50 values for the peptides studied are given in Table 2.

Figure 4d shows the Hill plot for the data given for bovine /I-endorphin in Fig. 2a. The slope of the plot is 0.92, i.e. close to 1.0 indicating that a cooperative in- teraction of /I-endorphin with the AChE was not tak- ing place.

DISCUSSION

The results presented describe the inhibition of acetylcholinesterase by a series of peptides which occur in the rat CNS and blood,14*“1 and which are biochemically-unrelated to acetylcholine or to the products of its hydrolysis. The results show that the inhibitory action of @ndorphin on acetylcholin- esterase is restricted to the 16s oligomer which is located, on the skeletal muscle surface membrane, ex- clusively in the vicinity of the motor endplate.7*s*26 It has been reported that the activity of the 16s form of the enzyme declines selectively following denerva- tion.6*26 In this laboratory we have shown a decrease of approximately 60% in the activity of 16s AChE in hind-limb muscles 45 days after section of the sciatic

Table 2.

Peptide I.C.50

Porcine /I-endorphin 2.1 + 1.8nM Bovine j?-endorphin 2.4 rf: 1.6nM C’,,-Fragment >O.l M C’,,-Fragment 0.4 + 0.36 PM /I-lipotropin 8.1 f 1.4nM

Concentrations of peptides producing 50% inhibition of 16s acetylcholesterase. Mean f S.E.M. calculated from 3-5 esti- mations.

nerve (L. W. Haynes, A. J. Harborne & M. E. Smith, unpublished observation). The finding that /I-endor- phin reduced the activity of AChE only in the plasma membrane subcellular fraction and that the inhibition was not as marked with the fraction prepared in the same way from denervated muscle is consistent with a selective action of the peptide on the 16s oligomer.

/3-Endorphin was the most potent of the peptides tested as an inhibitor of 16s AChE, whilst /I-lipotro- pin, followed by its C-fragment were less potent. Methionine enkephalin was a very much weaker in- hibitor than all of the larger peptides. Met-enkephalin and C-fragment, both contain the 61-65 opiate receg tor recognition sequence of fl-lipotropin1s*2g but lack the 88-91 C terminus. The relatively weak actions of these two peptides compared to those of /I-lipotropin and fl-endorphin indicate that the recognition sites for 16s AChE inhibition may not be identical to that of the classical opiate receptor. It is possible that the C terminal part of the /I-lipotropin and B-endorphin molecules is important for full inhibitory potential to be expressed. The importance of the lipotropin C ter- minal sequence in the enzyme inhibition is also con- sistent with the observation that bovine j?-endorphin (with a similar amino acid sequence to rat b-endor- phin,‘7.23 ) produced a greater maximum inhibition of AChE than porcine /l-endorphin, since the two com- pounds differ only at the C terminus in the amino acid at position 83.’ The finding that j?-lipotropin also inhibited 16s AChE was unexpected since this peptide has no analgesic or behavioural activity at physiological doseP3 and shows insignificant bind- ing to opiate receptors.3’ However, its activity as an enzyme inhibitor could be more easily understood if there is a recognition site for the inhibition of 16s AChE which is separate from that for the opiate receptor. The effect of naloxone is also consistent with this idea; naloxone only very weakly prevented the inhibition of 16s AChE in the presence of B-endor- phin although it is a powerful opiate antagonist.

1012 L. W. Haynes and M. E. Smith

Possible mechanism of action of /?-endorphin

It is interesting that only the high molecular weight 16s form of AChE is inhibited by these peptides since all the active sites in the high-molecular weight forms of the enzyme are identica12’ Tensilon, a weak com- petitive inhibitor of AChE, 3o decreased the activity of all three major high molecular weight forms. The in- hibition of AChE by endorphins may therefore not involve an interaction with catalytic sites, but may depend on the binding of the peptide inhibitors with a different site found only in the 16s oligomer. Sub- strate inhibition, an allosteric effect, has also been found to influence most markedly the 16s peak of activity in separated rat muscle AChE.’ Supporting evidence for the presence of a separate regulatory site for endorphin binding is provided from studies on the effect of digestion of the enzyme with collagenase on the inhibitory action of /3-endorphin. The procedure was designed to degrade the collagen attachment sub- units of the molecule.3 After the digestion, the cata- lytic activity of the enzyme was unchanged, but it was no longer inhibited by ,!?-endorphin. The inhibitory action is therefore probably not a direct action on the catalytic site. It may depend on the association of the enzyme with its collagen tails being preserved and involve a site on the collagen subunit. The slope of the Hill plot of inhibition data for fl-endorphin at different concentrations indicated that the binding of the inhibitor was not cooperative. Studies on the inhi- bition by /?-endorphin of AChE at different substrate concentrations were consistent with the type of inhi- bition being allosteric. Met-enkephalin, on the other hand, inhibited the enzyme activity in a non-competi- tive manner and also inhibited the enzyme following its digestion with collagenase. The retention of /3-endorphin in the 16s enzyme fraction after separ-

ation on sucrose density gradients may indicate that it may co-separate by virtue of the fact that it is bound to the enzyme, in which case the binding may only be slowly reversible. At present, we have no further data regarding the nature of the inhibitory binding site. If a regulatory subunit is present in the 16s AChE mol- ecule it may be possible to identify it in binding studies with radioactively-labelled peptides.

Conclusions

The results clearly demonstrate the selective inhi- bition of the 16s form of acetylcholinesterase in rat skeletal muscle by fl-endorphin and related naturally- occurring peptides. Previous work has demonstrated the presence of one or more of these peptides in the motor nerve axons of the rat during a period of devel- opment. l2 It is not known whether these peptides are released at the motor endplate in immature rats, but this problem is currently being investigated in this laboratory. Preliminary experiments’ have shown that /I-endorphin increases the contracture response to acetylcholine of denervated rat skeletal muscles. The present results showing its inhibitory action on AChE provide an explanation for this effect. The physiological significance of these findings remains to be elucidated. The inhibitory effect of B-endorphin is restricted to a form of the enzyme found chiefly at peripheral sites2’ and it would therefore be interesting to investigate its clinical potential as an anticholin- esterase drug devoid of any central actions.

Acknowledgements-We wish to thank Dr A. J. Harborne for performing denervations, and to acknowledge the sup- port of the Medical Research Council and the National Fund for Research into Crippling Diseases.

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(Accepted 13 December 1981)

MC. 714-K