Neuroscience Letters, 116 (1990) 29 33 Elsevier Scientific Publishers Ireland Ltd.

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Opioid receptors in skeletal muscle of normal and dystrophic mice S h a r o n H u g h e s a n d M a r g a r e t E. S m i t h Department of Physiology, Medical School, University of Birmingham, Birmingham ( U.K.) (Received 26 February 1990;Revisedversion received 17 April 1990;Accepted23 April 1990) Key words." Opioid receptors; fl-Endorphin; Muscular dystrophy; Skeletal muscle; Naloxone; MDX mouse

Autoradiography was used to investigate the presence of opioid receptors in soleus, extensor digitorum longus (EDL) and diaphragm muscles in two strains (C57BL/6J and C57BL/10) of mice whichcan inherit muscular dystrophy. Bindingsites for two radiolabelled opioid ligands [125I]fl-endorphinand [3H]naloxone, were seen in a few muscle fibres in the normal mice. However, a significantly greater number of fibres exhibited the binding sites in the dystrophic individuals of both strains. The binding sites were not restricted to the endplate regions but were present over the entire surface in these fibres.

fl-Endorphin immunoreactivity has been detected in motor nerve terminals at muscle endplates in rats and mice [7, 13]. It is present at a few endplates in normal adult mice but the incidence of immunoreactive endplates is higher in developing mice [7, 14]. fl-Endorphin can be released from immature nerve-muscle preparations upon stimulation of the nerve [8] and roles for this peptide at the neuromuscular junction have been suggested [3, 6, 9]. Muscular dystrophy is a disease characterised by delayed development of the neuromuscular system [4, 16]. In murine dystrophy there is a high incidence of fl-endorphin-immunoreactive motor nerves throughout life [7, 12]. In this study we have investigated the presence offl-endorphin receptors in muscle fibres of mice of the C57BL/10 and C57BL/6J strains which can inherit muscular dystrophy. In the C57BL/10 strain the genetic defect in the diseased (MDX) individuals is the same as in human Duchenne dystrophy and the gene product, dystrophin is absent in both the affected human and the M D X mouse [11]. However, the muscle weakness is progressive in the human but is only apparent for a restricted time during development in the M D X mouse [1, 5]. In the C57BL/6J mice the genetic defect is different but the disease follows a progressive course.

Correspondence." M.E. Smith, Department of Physiology, Medical School, University of Birmingham, Birmingham, B15 2TT, U.K. 0304-3940/90/$ 03.50 © 1990Elsevier Scientific Publishers Ireland Ltd.

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Fig. l. Opioid binding on skeletal muscles. A: transverse section of an EDL muscle of an M D X mouse after incubation with [~25I]/?-endorphin. B: longitudinal section of diaphragm muscle of a dystrophic C57BL/6J mouse, after incubation with pH]naloxone. Binding of the radiolabelled ligand is evident around the surface membrane of some fibres (arrows) and an endplate region (arrowheads) is shown in B. Bars = 20 ,urn.

31 Autoradiography was used to demonstrate the presence of ~-endorphin receptors in skeletal muscles from normal and genetically abnormal mice o£ the two strains. Mice of 10 weeks of age were killed by anaesthetisation with diethylether. The muscles were carefully dissected out and washed thoroughly in 0.1 M PBS (pH 7.4), and then rapidly frozen (slightly stretched) in isopentane, cooled in liquid nitrogen. Twenty-pm Cryostat sections were prepared and placed on subbed slides. The sections were stained for acetylcholinesterase activity using the procedure of Pestronk & Drachman [15] to localise the endplate regions. Autoradiography was carried out by a method based on that of Herkenham and Pert [10]. The slides were then washed by incubation in Tris buffer (0.17 M, pH 7.4), then in Tris buffer containing 0.5 mM GTP. They were then incubated for 60 min in a solution of either [125IXB-endorphin (10 nM, 2000 Ci/mmol) or [3H]naloxone (1 nM, 49.4 Ci/mmol) in Tris buffer (0.17 M, pH 7.4). Control sections were incubated in the appropriate solution of radioactive ligand but with a 100-fold excess of the unlabelled compound, to detect nonspecific staining. After incubation the sections were washed in Tris buffer (3 x 5 min) and then in distilled water for 5 min. After drying the sections were fixed over paraformaldehyde vapour at 80°C for 2 h to cross-link the ligand-receptor complexes and left overnight at room temperature to remove excess formaldehyde. The slides were then treated with K5 nuclear emulsion (Ilford), the exposure time being 1 week and 12 weeks (at - 2 0 ° C ) for sections incubated with [125I]fl-endorphin and [3H]naioxone, respectively. They were developed in D19 developer (Kodak), and counter-stained with Mayers haemalum (BDH). [125I]fl-Endorphin binding sites were evident on the surface of some fibres of the different muscles in both the normal and mutant individuals in both strains of mouse. No concentration of binding sites was seen at the endplate regions of these fibres (see Fig. 1B). Fig. 1A shows a transverse section of an extensor digitorum longus (EDL) muscle from an MDX mouse where specific binding can be seen on the surface of some fibres. Table I compares the incidence of fibres exhibiting the binding sites in the muscles of normal and affected individuals. The results show that there were more fibres exhibiting the binding sites in the muscles from mutant individuals of both strains than in the respective muscles from the normal individuals. Significantly more fibres of the soleus and EDL muscles exhibited the binding sites in the C57BL/10 mice than in the C57BL/6J mice, and this was true for both the normal and the affected individuals. The results for the diaphragm muscles were similar in the normal mice of the two strains and also in the mutant mice in the two strains. In the case of the normal individuals of both strains and the MDX mice, binding sites could not be detected at any location other than the fibre surface membrane, but in the dystrophic C57BL/6J mice there was some localisation of binding to discrete areas of the endomysial connective tissue. In order to see whether the binding sites could be opioid receptors muscle sections were incubated with a radiolabelled opioid antagonist, [3H]naloxone. Fig. 1B shows a longitudinal section of a diaphragm muscle from a dystrophic C57BL/6J mouse after incubation of the section with [3H]naloxone. Table I shows the results obtained after incubation of sections with [3H]naloxone, for diaphragm, soleus and EDL mus-

32 TABLE I COMPARISON OF [*25I]fl-ENDORPHINAND [3H]NALOXONE BINDING IN NORMAL AND DYSTROPHIC MICE OF TWO STRAINS Results are given as the mean percentage of fibres per muscle which exhibit surface binding sites. The fibres were counted in longitudinal sections prepared from the central regions of the muscle (10-12 sections per muscle). Values are given as the means_+S.E.M. for 4 muscles in each case. P is the probability level determined using Student's t-test. C57BL/6J mice

C57BL/lO mice

Normal

Dystrophic P

Normal

A [~25I]fl-endorphin-labelledfibres Soleus 6.8_+2.3 EDL 5.7+1.5 Diaphragm 6.3_+3.4

33.0_+2.6

Opioid receptors in skeletal muscle of normal and dystrophic mice.

Autoradiography was used to investigate the presence of opioid receptors in soleus, extensor digitorum longus (EDL) and diaphragm muscles in two strai...
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