Conversion of dynorphin-(1-9) to [Leu5]enkephalin by the mouse vas deferens in vitro

European Journal of Pharmacology, 116 (1985) 159-163

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Elsevier

C O N V E R S I O N O F D Y N O R P H I N - ( I - 9 ) T O [LeuS]ENKEPHALIN BY T H E M O U S E VAS D E F E R E N S IN V I T R O LYNNE MILLER, MICHAEL J. RANCE, JOHN S. SHAW and JOHN R. TRAYNOR * Bioscience Department I1, IC1 Pharmaceuticals Division, Alderley Park, Macclesfield, Cheshire, and * Department of Chemistry, University of Technology, Loughborough, Leicestershire LE11 3TU, U.K.

Received 21 March 1985, revised MS received 25 June 1985, accepted 5 July 1985

L. MILLER, M.J. RANCE, J.S. SHAW and J.R. TRAYNOR, Conversion ofdynorphin-(1-9) to [LeuS]enkephalin by the mouse vas deferens in vitro, European J, Pharmacol. 116 (1985) 159-163. The inhibitory action of dynorphin-(1-9) on the electrically stimulated mouse vas deferens was seen to be antagonised by the ~-selective opioid antagonist ICI 174864. The observed 6-receptor mediated responses were partially, but not totally, prevented by peptidase inhibitors which protect the C- and N-termini of dynorphin-(1-9). [3H]Dynorphin-(1-9) is rapidly degraded by slices of vasa deferentia of the mouse. The major product of this metabolism co-elutes with [LeuS]enkephalin on reverse phase HPLC. It is concluded that a major component of the inhibitory effects of dynorphin-(1-9) on the mouse vas deferens is mediated by degradation to [Leu 5]enkephalin which in turn acts through 8-receptors. It is possible that in other in vitro and in vivo systems, the effects produced by dynorphin-(1-9) might be similarly mediated by 8-receptor activation. Dynorphin-(1-9)

[LeuS]enkephalin

8-Receptors

1. Introduction The dynorphins are a family of opioid peptides found throughout the CNS (Weber et al., 1982). Though previous work has shown that dynorphin(1-9) is highly selective for the x-opioid receptor (Corbett et al., 1982), these peptides are C terminal extensions of the rather 8-selective [LeuS]en kephalin (Fischli et al., 1982) and as such it is possible that the dynorphins could act as precursors for [LeuS]enkephalin, thus providing another transmitter with a different selectivity of action. In support of this hypothesis the opioid peptide [Met 5]enkephalyl-Arg6-Phe 7 is degraded to [MetS]enkephalin (Benuck et al. 1981; Yang et al., 1983) and enzymes have been described which convert [Met 5] and [LeuS]enkephaly]-Arg 6 to [MetS] - and [LeuS]enkephalin (Hook et al., 1982; * To whom all correspondence should be addressed: Department of Chemistry, University of Technology, Loughborough, Leics. LEll 3TU, U.K. 0014-2999/85/$03.30 © 1985 Elsevier Science Publishers B.V.

Metabolism

Mouse vas deferens

Fricker and Snyder, 1982) and may be associated with enkephalin biosynthesis. In the mouse vas deferens it has been suggested that dynorphin-(1-8) may act via 6-receptors as determined by the effectiveness of the non-specific antagonist MR2266 to block its action (Oka et al., 1983). In this paper we confirm this for dynorphin-(1-9) using the highly 8-selective antagonist N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI 174864) (Cotton et al., 1984) and demonstrate that this is due to conversion of dynorphin-(1-9) to [LeuS]enkephalin in this tissue.

2. Materials and methods 2.1. Pharmacological assays

Vasa deferentia from mice of the Alderley Park strain were mounted in 5ml organ baths containing Krebs-Henseleit solution, from which magnesium was omitted, at 37°C and aerated with

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95% O2-5~ CO 2. The tissues were stimulated by trains of pulses (1 ms pulses at 50 Hz for 100 ms: supramaximal voltage) at 10 s intervals. Twitch heights were recorded isotonically under a tension of 200 mg. Cumulative dose-response curves were obtained to agonists in the absence and presence of ICI 174864 (5 /~M) and dose ratios were determined. From these results apparent K~ values were calculated as K¢ = [antagonist]/(dose ratio 1). To investigate the effects of peptidase enzyme inhibition, bestatin (30 #M), thiorphan (0.3 /~M), captopril (10 /~M) and L-leucyl-L-leucine (2 mM) were added to the Krebs solution (McKnight et al, 1983).

2.2. Metabolism studies Vasa deferentia from mice of the Alderley Park strain were sliced (100 /~m) and placed in 5 ml Mg2+-free Krebs solution at 37°C in the absence or presence of bestatin (30 /~M), thiorphan (0.3 /~M), captopril (10 /~M) and L-leucyl-L-leucine (2 raM). [3H]Dynorphin-(1-9) was added to give a final concentration of 100 nM. After 2.5 min an aliquot (400 /~1) was removed, centrifuged at 13 000 × g for 1 rain and the supernatant quickly frozen in liquid nitrogen and then stored at - 20°C prior to HPLC separation. To the above supernatants (100/~1) were added the marker peptides HTyrOH, HTyr-GlyOH and HTyr-Gly-GlyOH (3 ng); HTyr-Gly-GIy-PheOH, [LeuS]enkephalin, [LeuS]enkephalyl-Arg 6, [LeuS]enkephalyl-Arg6-Arg 7, dynorphin-(1-8) and dynorphin-(1-9) (100 ng). The samples were applied to an Altex Ultrasphere ODS column (150 mm × 4.6 mm, particle size 5/xM) and eluted with buffer consisting of 50 mM monosodium phosphate, 1 mg. ml 1 phosphoric acid and 5% MeOH, pH 2.7 containing 17% acetonitrile. The flow rate was 1 ml. min- 1. The various fractions were identified by electrochemical detection: the eluate from the column was passed through a null volume glassy carbon detector cell, with an applied potential of 1.0 V, before being collected in 1 ml fractions for scintillation counting. This system did not separate HTyr-Gly-Gly-PheOH from [Leu 5] enkephalyl-Arg6-Arg 7 or H T y r O H from HTyrGlyOH and HTyr-Gly-GlyOH. These components

were separated by re-chromatographing the relevant fractions under similar conditions but with a reduced level of acetonitrile (12%) or in the absence of acetonitrile respectively.

2. 3. Peptides and drugs [3H]Dynorphin-(1-9) (26 Ci. mmol l) was obtained from Amersham International. Dynorphin(1-9), [D-Thr2,L-LeuS]enkephalyl-Thr6, N,N-diallyl-Tyr-Aib-Aib-Phe-LeuOH (ICI 174864) and tifluadom were provided by Chemistry Dept. II, 1CI Pharmaceuticals Division. Other materials were obtained as follows. HPLC standards: HTyrOH, HTyr-GlyOH, HTyr-Gly-GlyOH, HTyr-Gly-GlyPheOH, [LeuS]enkephalyl-Arg6-Arg 7, (Sigma Chemical Co.); [LeuS]enkephalin, [LeuS]enke phalyl-Arg 6, dynorphin-(1-8) (Cambridge Research Biochemicals). Peptidase inhibitors: captopril (Squibb), thiorphan and L-leucyl-L-leucine (Chemistry Dept. II, ICI Pharmaceuticals Division), bestatin (Cambridge Research Biochemicals).

3. Results

3.1. Antagonist activity of IC1 174864 against dynorphin-(1-9) The depressant effect of dynorphin-(1-9) on the electrically induced contractions of the mouse vas deferens was antagonised by the &antagonist ICI 174864 with an apparent K e of 336 nM (table 1). This value corresponds to neither the ~- nor the &affinity of the antagonist, but is intermediate between the two values indicating that dynorphin(1-9) probably exerts its effect at both receptor types. To determine whether the observed &receptor mediated effects were due to metabolism of dynorphin-(1-9) to more &selective components, the assays were repeated in the presence of the peptidase inhibitors bestatin (30 /~M), thiorphan (0.3 /~M), captopril (10 /~M) and L-leucyl-L-leucine (2 mM) as recommended by McKnight et al. (1983). The mixture of inhibitors significantly reduced the potency of ICI 174864 in antagonising the action

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of dynorphin-(1-9) (K e 3150 nM). The fact that antagonism was still seen suggests a substantial ~-component, even in the presence of the inhibitors.

A

3.2. Metabolism of [~H]dynorphin-(1-9) Dynorphin-(1-9) and its metabolites were eluted from the reverse phase HPLC column using an ±socratic gradient of phosphate buffer pH 2.7, methanol (5%) and acetonitrile (17%) (fig. 1). The peaks were identified by electrochemical detection of the tyrosine moiety. The system did not separate HTyrOH from HTyr-GlyOH and HTyrGly-GlyOH, nor HTyr-Gly-Gly-PheOH from [Leu~]enkephalyl-Arg6-Arg7. These could however be separated using a less hydrophobic buffer system (fig. 1B,C). [3H]Dynorphin-(1-9) was rapidly broken down by mouse vasa deferentia slices• After 2.5 min the level of [3H]dynorphin-(1-9) was reduced to 16% and all possible breakdown products with the exception of the dipeptide HTyr-GlyOH were observed (table 2). The major metabolite (32%) eluted with the [Leu~]enkephalin marker. This fraction also proved to be relatively stable and after 10 min incubation it was still the major component (42.69 + 1.3%, n = 3) whilst [3H]dynorphin-(1-9) was reduced to 2.77 + 1.4% (n = 3). The breakdown was enzymic since using heattreated tissue (95°C, 15 min) no detectable alteration in the [3H]dynorphin level was observed. TABLE 1 Antagonist potencies of N,N-diallyI-Tyr-Aib-Aib-Phe-LeuOH (1CI 174864) on the isolated vas deferens of the mouse in the absence and presence of peptidase inhibition. Opiate

K~ (nM) Alderley Park mice Control

Dynorphin-(1-9) [D-Thr 2 L-Leu 5 ]enkephalyl-Thr 6 Tifluadom

336.6 ± 100.4 (8) 30.6 _+7.0 (4) > 5000 (8)

With peptidase inhibition * 3150

+853(4)

29:9 ± 6.3 (4) > 5000 (4)

* The Krebs solution contained the following peptidase inhibitors: bestatin (30 btM), thiorphan (0.3 /~M), captopril (10 /~M) and L-leucyl-L-leucine (2 raM). Bath temperature was 37°C. The values are the m e a n s ± S . E M . The number of observations is given in parentheses.

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Fig. 1. Typical HPLC separation of dynorphin-(1-9) and its metabolites after 2.5 rain contact with vasa deferentia slices using an Altex uhrasphere ODS column (150 m m × 4 . 6 mm, particle size 5 /~m). Arrows indicate positions of marker standards determined with electrochemical detection. (A) Separation using buffer consisting of 50 m M monosodium phosphate, 1 m g - m l - I phosphoric acid, 5% methanol pH2.7 with 17% acetonitrile; (B) further• separation of peak 2 (HTyr-GlyGly-PheOH, [LeuS]enkephalyI-Arg 6 using above buffer but 12% acetronitrile; (C) further separation of peak 1 (HTyrOH; HTyr-GIyOH; HTyr-GIy-GlyOH) using above buffer in absence of acetonitrile. In all cases the flow rate was 1 m l . m i n 1 and I ml fractions were collected for scintillation counting.

TABLE 2 Distribution of 3H-label between dynorphin-(1-9) and its metabolites after 2.5 min contact with slices of vasa deferentia from the mouse. Metabolite (order of elution)

HTyrOH HTyr-GIy-GIyOH HTyr-GIy-OH HTyr-GIy-GlyPheOH Leu-enkephalyl-Arg6-Arg7 Leu-enkephalyl-Arg 6 Dynorphin-(1-9) Leu,enkephalin Dynorphin-(1-8)

% recovery Control 14.17 __.1.52 8.48 ___1.11 N.D. 9.20 ___2.76 9.85 ___2.32 9.51 ± 0.77 16.37±5.17 32.43 ± 1.85 0.33 + 0.09

Peptidase inhibitors * 9.92 + 0.54 2.13+0.15 21.66 + 0.45 29.94+_2.11 36.08 +_1.24 0.25 _+0.12

* As in table 1, N.D. not detected. Values are means + S.E.M. from three separate experiments. At zero time [3H]dynorphin-(1-9) purity was 91.82+ 1.36% (n = 3). The major impurity (6.35 + 1.16% n = 3) eluted in the fraction containing HTyrOH; HTyr-Gly-GlyOH; HTyr-GIyOH.

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In the presence of peptidase inhibitors (see table 1) the [3H]dynorphin-(l-9) was stabilised. However the inhibitors did not appear to prevent the production of [LeuS]enkephalin, or [LeuS]en kephalyl-Arg 6.

4. Discussion

The results demonstrate that the •-ligand dynorphin-(1-9) is converted by the mouse vas deferens to the more stable, &preferring [LeuS]enkephalin. A &component of the pharmacological action o f dynorphin-(1-9) can be readily demonstrated by use of the antagonist ICI 174864 which is highly selective for the 6-receptor (Cotton et al., 1984), in contrast to naloxone or MR2266 which show relatively poor discrimination between ~¢- and a-receptors. The metabolism studies show that the addition of a mixture of peptidase inhibitors to protect the N- and C-termini of dynorphin(1-9) (McKnight et al., 1983) did increase the overall stability of dynorphin-(1-9). However, this was not at the expense of conversion to the more 8-selective peptides [LeuS]enkephalin and [LeuS]enkephalyl-Arg 6. This was confirmed by the pharmacological studies as antagonism of dynorphin-(1-9) by ICI 174864 was still observed at < 5 ~M in the presence of peptidase inhibition, although this latter result could be due to a small inherent affinity of dynorphin-(1-9) for the 6-receptor. Since the peptidase inhibitors utilised failed to prevent [Leu5]enkephalin production there is a possibility that the dynorphin-(1-9) is cleaved directly at the LeuS-Arg 6 bond to yield [LeuS]enkephalin and at the Arg6-Arg 7 bond to yield [LeuS]enkephalin-Arg 6. Recently Fricker and Snyder (1983) reported an enzyme which selectively degrades hexapeptides and heptapeptides containing a C-terminal basic amino-acid residue and which may be important ih providing [LeuS]enkephalin in brain and adrenal gland. However, the amount of [LeuS]enkephalyl-Arg 6Arg 7 produced in the above assay containing the peptidase inhibitor cocktail is very low, suggesting

that captopril and L-leucyl-L-leucine are protecting the C-terminus from attack. Nevertheless we cannot rule out a rapid turnover of the [LeuS]enkephalyl-Arg6-Arg 7 moiety. The production of [LeuS]enkephalin from dynorphin-(1-9) has important implications. Firstly, the metabolism to [LeuS]enkephalin must be considered as a conversion, rather than an inactivation process. This raises the question of the physiological relevance of our observations which may suggest that in the vas deferens and possibly at other sites, [LeuS]enkephalin might be formed from pro-dynorphin, in addition to, or rather than, pro-enkephalin. As far as we are aware, the presence of dynorphins in the vas deferens has not been demonstrated, but it is possible that variable post-translational processing could occur from the large, stable dynorphin-(1-17) to the short acting &agonist [LeuS]enkephalin, as postulated by Hughes (1983). Alternatively the enkephalin might be processed directly from prodynorphin as may be the case in the porcine neurointermediate lobe (Kilpatrick et al., 1983) and in the heart (Weihe et al., 1985). On the other hand, dynorphin and enkephalin systems are anatomically separate in brain and prodynorphin is unlikely to be a major source of [LeuS]enkephalin in this tissue (Watson et al., 1982), although Zamir et al., (1984) have provided evidence to suggest [LeuS]enkephalin levels in the rat substantia nigra may be generated from prodynorphin. It would be interesting to know how our observations relate to the action of dynorphins in central tissues and, in particular, to the spinal actions of dynorphin-(1-8). The effects we have observed, particularly with the slice preparation, including both the pattern of dynorphin-(1-9) metabolism and the degree to which metabolism occurs, may well be due to non-specific endopeptidases and not at all associated with neuronal elements in the intact tissue. However, it is still important to note that, even in the presence of peptidase inhibition, a considerable component of the effects of the 'small' dynorphins may be mediated by action at the 8receptor and therefore caution must be exercised in the interpretation of results.

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