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Autoradiographic localization of δ-opioid binding sites in human sigmoid colon
European Journal of Pharmacology, 142 (1987) 185-186
185
Elsevier
EJP 085RC
Rapid communication
Autoradiographic localization of 8-opioid binding sites in human sigmoid colon S. J a m e s 1, C.H.V. H o y l e 1, (3. B u r n s t o c k 1.., J.R. Jass 2, I.J.M. J e f f r e y 2 and J.E. L e n n a r d - J o n e s 2 I Department of Anatomy and Developmental Biology, and Centre for Neuroscience, University College London, Gower Street, London WCIE 6BT, and e St Marks Hospital, City Road, London ECI, UK. Received 1 September 1987, accepted 3 September 1987
Studies on the rabbit colon in vivo have shown that enkephalins have a depressant effect on inhibitory neuromuscular transmission in both the distal and proximal colon, and a facilitatory action on excitatory neuromuscular transmission in the proximal colon (Blanquet et al., 1982). These results suggest a modulatory role for enkephalins on intestinal motility. In the present study, we used the technique of in vitro labelling and autoradiography to directly examine the localization of 8-opioid receptors in the myenteric plexus and muscularis of the human colon using [ 3H][D-AIa2,D-Leu5 ]enkephalin (DADL). This is a stable analogue of [LeuS]en kephalin and is regarded as a selective opioid receptor agonist (Wuster et al., 1979). Segments of uninvolved human distal sigmoid colon were obtained from fresh surgical specimens removed for rectal carcinoma from three women and two men aged 62-77 years. The segments were placed on a cork block, coated with plastic embedding medium (OCT Compound Lab-Tek Products) and frozen in liquid nitrogen. Cryostat sections (18 btm) were cut at - 1 6 ° C, thaw-mounted onto uncoated microscope slides and kept under vacuum at 4 ° C until dry. Slides were preincubated for 15 min in 50 mM Tris-HC1, pH 7.4, followed by an incubation of 30 rain in the same buffer with the addition of 24 nM [3H][D-Ala2,D-
* To whom all correspondence should be addressed.
LeuS]enkephalin (DADL; 30-50 Ci/mmol, New England Nuclear). Non-specific binding was determined by incubating sequential sections in the same media but in the presence of unlabelled ligand (DADL; Cambridge Research Biochemicals) at a concentration of 20 ~tM. All incubations were performed at room temperature. Sections were washed for 2 × 30 s in ice-cold buffer and 10 s in ice-cold distilled water. The sections were then dried using a forced stream of filtered, cold, dry air before apposition to emulsion-coated (Ilford K2) acid-washed glass coverslips. After 4-6 weeks of exposure at 4 ° C, autoradiographs were developed in Kodak D19 and the sections stained with toluidine blue and mounted for a semi-quantitative light microscopic analysis. Grain counting was performed by use of a microscope equipped with a calibrated eye-piece. The distribution of [3H]DADL is shown in fig. 1. The greatest density of binding was associated with neurones of the myenteric plexus situated between the longitudinal and circular muscle layers. About 80-90% of the neurones are labelled. Generally, a low density of labelled receptors was found on the muscle layers, with the circular muscle exhibiting approximately 20% more labelling than the longitudinal muscle. In control sections, incubated in the presence of unlabelled DADL, the density of grains was greatly reduced in all densely labelled regions. The 8-opioid receptors visualized in the muscle layers of the human colon may be located on the
0000-0000/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
186
non-adrenergic, non-cholinergic inhibitory nerve fibres innervating the smooth muscle. In preparations of circular muscle devoid of myenteric plexus, activation of these receptors by enkephalins causes prejunctional inhibition of non-adrenergic, noncholinergic inhibitory neurotransmission without having any direct action on the smooth muscle (Hoyle et al., 1986). The receptors seen in the myenteric plexus are present on ganglionic neurones which may well receive an input from enkephalinergic nerves, especially since extensive networks of fibres containing immunoreactivity to enkephalin have been observed in the myenteric plexus of the human gut (Polak et al., 1977). This work adds further support for enkephalins having a role in the modulation of colonic motility.
References
Fig. 1. Autoradiographical localization of D A D L receptor binding sites in the h u m a n myenteric plexus and muscularis. (A) is a light-field photomicrograph of a transverse section of h u m a n myenteric plexus and muscularis stained with tohiidine blue, while (B) is a dark-field photomicrograph of the emulsion-coated coverslip which overlaid (A) for 5 weeks. Notice that most binding sites are over the myenteric plexus neurones found within the ganglia (mg), with a low density over the circular muscle (cm) and longitudinal muscle (lm). Note that not all the neurones within a given ganglion are always labelled (*). When excess unlabelled D A D L was included in the incubation medium, specific binding over both the muscularis and the ganglia was greatly reduced. Scale bar = 100 g m .
Blanquet, F., M. Bouvier and J. Gonella, 1982, Effects of enkephalins and morphine on spontaneous electrical activity and on junction potentials elicited by parasympathetic nerve stimulation in cat and rabbit colon, Br. J. Pharmacol. 77, 419. Hoyle, C.H.V., G. Burnstock, J. Jass and J.E. Lennard-Jones, 1986, Enkephalins inhibit non-adrenergic, non-cholinergic neuromuscular transmission in the h u m a n colon, European J. Pharmacol. 131, 159. Polak, J.M., S.R. Bloom, S.N. Sullivan, P. Facer and A.G.E. Pearse, 1977, Enkephalin-like immunoreactivity in the hum a n gastrointestinal tract, Lancet 1,972. Wuster, M., R. Schulz and A. Herz, 1979, Specificity of opioids towards the ~-, 8- and c-opiate receptors, Neurosci. Lett. 15, 193.
185
Elsevier
EJP 085RC
Rapid communication
Autoradiographic localization of 8-opioid binding sites in human sigmoid colon S. J a m e s 1, C.H.V. H o y l e 1, (3. B u r n s t o c k 1.., J.R. Jass 2, I.J.M. J e f f r e y 2 and J.E. L e n n a r d - J o n e s 2 I Department of Anatomy and Developmental Biology, and Centre for Neuroscience, University College London, Gower Street, London WCIE 6BT, and e St Marks Hospital, City Road, London ECI, UK. Received 1 September 1987, accepted 3 September 1987
Studies on the rabbit colon in vivo have shown that enkephalins have a depressant effect on inhibitory neuromuscular transmission in both the distal and proximal colon, and a facilitatory action on excitatory neuromuscular transmission in the proximal colon (Blanquet et al., 1982). These results suggest a modulatory role for enkephalins on intestinal motility. In the present study, we used the technique of in vitro labelling and autoradiography to directly examine the localization of 8-opioid receptors in the myenteric plexus and muscularis of the human colon using [ 3H][D-AIa2,D-Leu5 ]enkephalin (DADL). This is a stable analogue of [LeuS]en kephalin and is regarded as a selective opioid receptor agonist (Wuster et al., 1979). Segments of uninvolved human distal sigmoid colon were obtained from fresh surgical specimens removed for rectal carcinoma from three women and two men aged 62-77 years. The segments were placed on a cork block, coated with plastic embedding medium (OCT Compound Lab-Tek Products) and frozen in liquid nitrogen. Cryostat sections (18 btm) were cut at - 1 6 ° C, thaw-mounted onto uncoated microscope slides and kept under vacuum at 4 ° C until dry. Slides were preincubated for 15 min in 50 mM Tris-HC1, pH 7.4, followed by an incubation of 30 rain in the same buffer with the addition of 24 nM [3H][D-Ala2,D-
* To whom all correspondence should be addressed.
LeuS]enkephalin (DADL; 30-50 Ci/mmol, New England Nuclear). Non-specific binding was determined by incubating sequential sections in the same media but in the presence of unlabelled ligand (DADL; Cambridge Research Biochemicals) at a concentration of 20 ~tM. All incubations were performed at room temperature. Sections were washed for 2 × 30 s in ice-cold buffer and 10 s in ice-cold distilled water. The sections were then dried using a forced stream of filtered, cold, dry air before apposition to emulsion-coated (Ilford K2) acid-washed glass coverslips. After 4-6 weeks of exposure at 4 ° C, autoradiographs were developed in Kodak D19 and the sections stained with toluidine blue and mounted for a semi-quantitative light microscopic analysis. Grain counting was performed by use of a microscope equipped with a calibrated eye-piece. The distribution of [3H]DADL is shown in fig. 1. The greatest density of binding was associated with neurones of the myenteric plexus situated between the longitudinal and circular muscle layers. About 80-90% of the neurones are labelled. Generally, a low density of labelled receptors was found on the muscle layers, with the circular muscle exhibiting approximately 20% more labelling than the longitudinal muscle. In control sections, incubated in the presence of unlabelled DADL, the density of grains was greatly reduced in all densely labelled regions. The 8-opioid receptors visualized in the muscle layers of the human colon may be located on the
0000-0000/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
186
non-adrenergic, non-cholinergic inhibitory nerve fibres innervating the smooth muscle. In preparations of circular muscle devoid of myenteric plexus, activation of these receptors by enkephalins causes prejunctional inhibition of non-adrenergic, noncholinergic inhibitory neurotransmission without having any direct action on the smooth muscle (Hoyle et al., 1986). The receptors seen in the myenteric plexus are present on ganglionic neurones which may well receive an input from enkephalinergic nerves, especially since extensive networks of fibres containing immunoreactivity to enkephalin have been observed in the myenteric plexus of the human gut (Polak et al., 1977). This work adds further support for enkephalins having a role in the modulation of colonic motility.
References
Fig. 1. Autoradiographical localization of D A D L receptor binding sites in the h u m a n myenteric plexus and muscularis. (A) is a light-field photomicrograph of a transverse section of h u m a n myenteric plexus and muscularis stained with tohiidine blue, while (B) is a dark-field photomicrograph of the emulsion-coated coverslip which overlaid (A) for 5 weeks. Notice that most binding sites are over the myenteric plexus neurones found within the ganglia (mg), with a low density over the circular muscle (cm) and longitudinal muscle (lm). Note that not all the neurones within a given ganglion are always labelled (*). When excess unlabelled D A D L was included in the incubation medium, specific binding over both the muscularis and the ganglia was greatly reduced. Scale bar = 100 g m .
Blanquet, F., M. Bouvier and J. Gonella, 1982, Effects of enkephalins and morphine on spontaneous electrical activity and on junction potentials elicited by parasympathetic nerve stimulation in cat and rabbit colon, Br. J. Pharmacol. 77, 419. Hoyle, C.H.V., G. Burnstock, J. Jass and J.E. Lennard-Jones, 1986, Enkephalins inhibit non-adrenergic, non-cholinergic neuromuscular transmission in the h u m a n colon, European J. Pharmacol. 131, 159. Polak, J.M., S.R. Bloom, S.N. Sullivan, P. Facer and A.G.E. Pearse, 1977, Enkephalin-like immunoreactivity in the hum a n gastrointestinal tract, Lancet 1,972. Wuster, M., R. Schulz and A. Herz, 1979, Specificity of opioids towards the ~-, 8- and c-opiate receptors, Neurosci. Lett. 15, 193.