Neuropeptides 5: 93-96, 1984

MU-RECEPTORS IN THE C57BL MOUSE VAS DEFERENS

Lynne Miller and J.S. Shaw

Bioscience Department II, ICI Pharmaceuticals Division, Alderley Park, Macclesfield, Cheshire SK10 4TG, U.K.

ABSTRACT

The C57 mouse vas deferens preparation has been shown to be insensitive to n-opioids. The present study indicates that this tissue possesses u, d and k-receptors with similar affinities to those found in other isolated tissue preparations, but has a small receptor reserve. Thus partial agonists such as morphine are detected as antagonists.

INTRODUCTION

Several workers have reported that mice of the C57 BL/6J strain (C57) are unusually insensitive to morphine. Oliverio and Castellano (1) observed that C57 mice were less sensitive to the antinociceptive effects of morphine than either the BALB/CJ or DBA/23 strains. Furthermore, these workers reported that tolerance to the effect of morphine develops more rapidly in C57 mice than in either of the other two strains studied. Similar results were obtained by Frigeni et al. (2) who reported that C57 mice could be rendered tolerant by a single dose of morphine.

A reduced responsiveness to morphine has also been observed in isolated vas deferens preparations (MVD) from C57 mice (3). The observations have since been extended by Waterfield et al. (4) who reported that although the C57 MVD exhibits a reduced sensitivity to morphine, it responds noneally to the enkephalins. It has thus been concluded that the C57 mouse either has a lower proportion of n-receptors (4) or that its morphine- sensitive receptors possess a lower affinity for the alkaloid (2).

The present study was designed to characterise the receptor s.ubtypes present in the C57 MVD with the aim of gaining some insight into the mechanism underlying the reduced efectiveness of morphine in this strain of mouse.

METHODS

Mice of the Alderley Park (AP) or C57BL/6J strains weighing between 25 and 309 were used throughout the study. Isolated vasa deferentia were

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prepared as previously described (5). Full cumulative dose-response curves were obtained for all agonists and from these IC5 concentrations were calculated by linear regression. Antagonists, w ere used, were allowed to fl equilibrate for 30 min before repetition of the agonist dose-response curve.

RESULTS

Figu e 1 sh ws dose-response curves for agonists (CD-Ala$,MePh 4 1 e ,G y-o15]enkephalin-GLYOL), d (CD-Thr

selecgive gor u ,Leu ,Thr ]

enkephalin-DTLET) and k (ethylketocyclazocine - EKC) receptors. It is clear that each agonist is more potent in the AP than in the C57 MVD, although the difference in potency is greater for GLYOL than for the other two agonists.

1 Fig

Dose-response curves to A) DTLET, 6) EKC and C) GLYOL in AP and C57 mouse vas deferens. Each

4 -7.5 -S.D -53 -1.5 point represents the mean of

KIG CONC~NTBITION at least 40 observations.

An indication of the receptor subtype at which each agonist produced its effert was obtained by determining the ability of the d-antagonist ICI 174864 (6) and naloxone to reverse the agonist effects. The Ke values obtained (Table 1) reveal little difference between the two strains of mice suggesting that the agonists and antagonists act at similar receptors in both preparations. Furthermore, the Ke values for naloxone against GLYOL suggest that this agonist acts at the p-receptor in both tissues.

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Table 1 The Antagonist Affinities of Naloxone and ICI 174864 against selective , d and k-agonists in C57BL and Alderley Park mice

Naloxone ICI 114864 AP C5/ AP c57

GLYOL 3.65 ? 0.42 (11) 4.44 -+ 2.17 (8) >5000 >5000 DTLET 29.9 f 4.1 (10) 22.1 + 3.53 (8) 32.9 -+ 4.7 ( ) EKC 15.5 f 2.1 (4) 17.9 f 4.2 (4) >5000

'".;,;,;.O (24)

All values are ke (nM) f s.e.m. Figures in parentheses indicate number of observations

Similarly, the Ke for naloxone against EKC and for ICI 1748964 against DTLET confirms the k and d-selectivity of these agonists.

An interesting point to emerge from this study was that morphine, which behaves an an agonist in the AP MVD, failed to depress twitch height in the C57 MVD. However, when tested as an antagonist in the C57 MVD, morphine was found to reverse the effects of GLYOL with a ke of 1.71 + 0.37ti (n=7).

Naloxone and EKC were gifts from DuPont and Sterling-Winthrop respectively.

DISCUSSION

It is clear from the results that the C57 MVD responds to GLYOL, DTLET and EKC. Furthermore, antagonist studies produced results which are consistent with these agonists acting at p, d and k-receptors respectively. There is thus no evidence for any difference in the affinities of the receptor populations in the two strains of mouse.

However, the C57 MVD is less sensitive to agonists and particularly to the P-agonist GLYOL. Furthermore, the C57 MVD fails to detect the agonist activity of morphine, although the ability of this compound to antagonise GLYOL clearly demonstrates that it possesses affinity for the p-receptor. Indeed, the afinity of morphine in this tissue is similar to that observed in another in vitro P-receptor model - the rat isolated vas deferens (7,8,9).

It would appear that, like the rat vas deferens, the C57 MVD has a small receptor reserve, and thus detects partial agonists as antagonists. The lower potency of the d- and k-receptor agonists suggests that this deficit in spare receptors applies to all the receptor subtypes present in the tissue.

Since it is likely that the process of tolerance involves a loss of spare receptors (lo), the finding that the C57 MVD possesses only a small receptor reserve could well explain the rapid aquisition of tolerance (1,2) observed in this strain.

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REFERENCES

1. Oliverio, A. and Castellano, C. (1974). Genotype-dependent sensitivity and tolerance tyo morphine and heroin: dissociation between opiate-induced running and analgesia in the mouse. Psychopharmacologia (Berl.) 39: 13-22.

2. Frigeni, V., Bruno, F., Carenzi, A. and Racagni, . (1981). Difference in the evelopment of tolerance to morphine and D-Ala 8 -methionine-enkephalin in C57BLl6J and DBAIZJ mice. Life Sciences 28: 729-736.

3. Henderson, G. and Hughes, J. (1976) The effects of morphine on the release of noradrenaline from the mouse vas deferens. Brit. J. Pharmacol. 57: 551.

4. Waterfield, A.A., Lord, J.A.H., Hughes, J. and Kosterlitz, H.W. (1978). Differences in the inhibitory effects of normorphine and opioid peptides on the responses of the vasa deferentia of two strains of mice. European J. Pharmacol. 47: 249-250.

5. Shaw, J.S. and Turnbull, M.J. (1978) In vitro profile of some opioid pentapeptide analogues. European J. Pharmacol. 49: 313-316.

6. Cotton, R.C., Giles, M.G., Miller, L., Shaw, J.S. and Timms, D. (1984) ICI 174864: A highly selective antagonist for the opioid d-receptor. European J. Phanacol. 97: 331-332.

7. Henderson, G., Robinson, D.S. and Sim, J.A. (1982) Antgonist actions of morphine on the rat vas deferens. Br. J. Pharmacol. 75 (Suppl) 29P.

8. Smith, C.F.C. and Rance, M.J. (1983) Opiate receptors in the rat vas deferens. Life Sciences 33 (Suppl. l), 317-320.

9. Carroll, J.A., Miller, L., Shaw, J.S. and Downes, C.P. (1984) Mu recceptor binding in physiological media: Comparison with isolated tissue data. INRC Proceedings.

10. Porreca, F. and Burks, T.F. (1983) Affinity of normorphine for its pharmacological receptor in the naive and morphine tolerant guinea-pig isolated ileum. J. Pharmacol. Exp. Ther. 225, 688-693.

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