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Comparison of opioid receptor distributions in the rat ileum
Life Sciences 78 (2006) 1610 – 1616 www.elsevier.com/locate/lifescie
Comparison of opioid receptor distributions in the rat ileum A.C. Gray, I.M. Coupar, P.J. White * Department of Pharmaceutical Biology and Pharmacology, Victorian College of Pharmacy, Monash University, 381 Royal Pde, Parkville, Victoria 3052, Australia Received 17 April 2005; accepted 26 July 2005
Abstract The cellular expression patterns of A-, y- and n-opioid receptors in the rat ileum were examined using fluorescence immunohistochemistry. Double-labelling was used to examine cellular receptor co-localisation as a pre-requisite for intracellular molecular interactions, such as heterodimerisation. Tissues were stained as whole-mount preparations. Strong, broadly distributed immunoreactivity (ir) was observed for each receptor in the myenteric and submucous plexuses. Although intracellular A- and y-ir patterns differed in ganglion neurons, A/y co-expression was extensive in these cells. A/y co-expression was also observed in interstitial cells, which were diffusely distributed in submucous plexus preparations but generally located adjacent to myenteric plexus structures. Punctate n-ir was seen broadly in nerve fibres in both plexuses, suggesting localisation in varicosities. Neuronal A/n co-localisation was not apparent, although n-ir fibres were often apposed against A-ir cells. A/ n co-localisation was detected in interstitial cells in submucous plexus preparations. Similarities in A and y expression patterns might reflect similar functional properties previously detected for these receptors. This study indicates that the rat gastrointestinal tract might provide a useful tool for the future study of molecular interactions between opioid receptor types. D 2005 Elsevier Inc. All rights reserved. Keywords: y-opioid receptor; Interstitial cells; n-opioid receptor; A-opioid receptor; Myenteric plexus; Submucous plexus
Introduction Opioid drugs strongly inhibit gastrointestinal transit, which, depending on the clinical circumstances, can be either deleterious (as a side-effect of opioid analgesic treatment) or beneficial (in the treatment of diarrhoea). Biochemical transduction of these effects involves molecular binding of the drug to opioid receptors located within the gut wall. The three wellestablished opioid receptor types, A, y and n, have each been shown to carry responsibility for opioid gastrointestinal effects in studies across various species. All three types are present in the intestinal wall of the rat (Bagnol et al., 1997; Dashwood et al., 1985; Fickel et al., 1997; Gray et al., 2005; Nishimura et al., 1986; Sternini et al., 1995). A and y receptor activation each lead to gastrointestinal transit arrest in the rat (La Regina et al., 1988; Tavani et al., 1984, 1990), and our group has recently
Abbreviations: A; A-opioid receptor; y; y-opioid receptor; n; n-opioid receptor; DMSO; dimethyl sulfoxide; FIT; Cluorescein isothiocyanate; ICC; interstitial cells of Cajal; ir; Immunoreactive (-ity); PBS; phosphate buffered saline. * Corresponding author. Tel.: +61 3 9903 9074; fax: +61 3 9903 9638. E-mail address: [email protected] (P.J. White). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.07.048
shown that these receptors mediate inhibition of electrically induced neurogenic smooth muscle contractions in the rat ileum (Gray et al., 2005). A receptors also appear to mediate anti-secretory effects (Coupar, 1983). Despite investigations into gastrointestinal transit (Tavani et al., 1984), muscle contractility (Gray et al., 2005), secretion (Coupar, 1983) and visceral antinociception (Su et al., 2002), the functional effects of rat enteric n receptor activation remain unclear. Immunohistochemistry has shown that A and n receptors are localised in enteric neurons and interstitial cells in the rat gut (Bagnol et al., 1997; Fickel et al., 1997; Sternini et al., 1995). The identity of these interstitial cells is not currently known. y receptors have only been localised histochemically using autoradiography, at considerably lower resolution (Nishimura et al., 1986). However, functional pharmacological evidence suggests neuronal localisation (Hancock and Coupar, 1994). Opioid receptors appear to possess the ability to form dimers and/or larger oligomers, with the effect of altering various receptor properties (Jordan and Devi, 1999). Effects may include pharmacological synergism, ligand affinity changes, cross-tolerance and cross-dependence. Heterodimerised opioid receptors may be responsible for the existence of pharmacologically definable opioid receptor types in the
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Table 1 Primary antibodies used for staining Molecular target
Host
Epitope sequence
Source
Dilution
A-opioid receptor (MOR-1)
Guinea-pig
NHQLENLEAETAPLP (C-terminal)
y-opioid receptor (DOR-1)
Rabbit
LVPSARAELQSSPLV (N-terminal)
Chemicon (Temecula, CA, USA), Cat. AB1774 Chemicon, Cat. AB1560
n-opioid receptor (KOR-1)
Rabbit
Internal region
Sigma, Cat. O1757
absence of single, isolatable corresponding genetic entities. An example is the y2 receptor, which is putatively dimerised A and y receptors (Gomes et al., 2000; Porreca et al., 1992; Xu et al., 1993). Better understanding of receptor oligomerisation may provide benefits to clinical drug use and drug development in the future. A logical requirement for oligomerisation is cellular co-localisation of the receptor proteins. Other potential intracellular interactions could occur at second messenger or subsequent levels, and would also necessitate co-localisation (Smith and Lee, 2003). It has been speculated that A and n receptors might be co-expressed by rat enteric neurons, since these receptors exhibit similar anatomical distributions, as revealed by immunohistochemistry (Fickel et al., 1997). Coexpression of A and n, and A and y receptors has already been demonstrated in guinea-pig ileum neurons using electrophysiological methods (Cherubini and North, 1985; Egan and North, 1981). Double-label immunohistochemistry has been used to show neuronal co-expression of y and n receptors in the pig ileum (Poonyachoti et al., 2001). The aim of this study was to establish whether the potential exists for intracellular and cellular opioid receptor interactions in the rat small intestine. Immunohistochemistry and doublelabel immunohistochemistry were used. The distributions of A,
1:200 – 1:500
1:500 – 1:1000
1:200 – 1:500
References (Pickel et al., 2004; Popper et al., 2004; Rodriguez et al., 2001) (Cahill et al., 2001; Commons and Milner, 1996, 1997; Persson et al., 2000; Varona et al., 2003) (Sigma-Aldrich, 2004)
y and n opioid receptors were examined, as was the extent to which cells express y and n receptors together with A receptors. Part of this study was presented at the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists (ASCEPT) meeting in 2003 (Gray et al., 2003). Materials and methods Animals and tissue preparation The tissue preparation method was similar to that of Costa et al. (1980). Three female Hooded Wistar rats (250 –300 g) (Victorian College of Pharmacy, Monash University, Australia) were heavily anaesthetised with thiopentone (120 mg/kg, i.p.), and transcardially perfused with 200 ml phosphate buffered saline, pH 7.4 (PBS), followed by 200 ml 4% formaldehyde in PBS. Terminal ileum segments (10 cm proximal to the caecum) were excised and thoroughly rinsed with PBS. The segments were sectioned along the mesenteric border and pinned flat and taut on balsawood boards (with the mucosa apposed), and postfixed in 4% formaldehyde in PBS overnight at 4 -C. The tissues were cleared with three 10 min washes in DMSO (as per the protocol of Grace and Llinas, 1985), which were followed
Fig. 1. Confocal micrographs showing the distribution of opioid receptor immunoreactivities in the myenteric plexus of the rat ileum. A, D: A-ir is visible in the cytoplasm of neuronal cell bodies in ganglia, but is weak or absent in fibres. Some lightly stained interstitial cells are located adjacent to plexus structures (indicated by arrowheads). An asterisk (*) indicates a neuron that has stained positive for A receptors, but not y receptors. B: y-ir extends to the tertiary plexus and is colocalised with A receptors in ganglia and interstitial cells. E: Punctate n-ir skirts ganglion cell bodies and extends into the tertiary plexus. A n-ir varicose fibre passes against a lightly-stained A-ir interstitial cell (indicated by an arrowhead) in the tertiary plexus. C, F: Overlaid images. Scale bar: 50 Am.
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by another set of PBS washes. The ileum wall layers were separated by manual microdissection to produce separate whole-mount preparations, enabling en face examination of the myenteric and submucous plexuses and adherent smooth muscle. Ethical approval was obtained from the Monash University Victorian College of Pharmacy Animal Experimentation Ethics Committee. All efforts were made to minimise the suffering and the number of animals used.
involving adjustments to contrast, brightness and gamma. Stained cells were counted manually to quantify the prevalence of receptor co-localisation. Staining controls, involving omission of either or both primary antibodies, were routinely prepared and imaged to establish specificity of staining. Levels of autofluorescence and non-specific secondary antibody binding were generally found to be very low.
Immunohistochemistry
Results
Preparations were incubated for 30 min in an antibody diluent solution, which contained 0.3% Triton X-100 (Sigma, St. Louis, MO, USA), 3% normal goat serum (Vector, Burlingame, CA, USA), 1% bovine serum albumin (Sigma) and 0.01% sodium azide (Sigma) in PBS. Following this, mixtures of primary antibodies labelling A and y, or A and n receptors (Table 1), were incubated with the free-floating preparations for 24 –48 h at 4 -C, in the same antibody diluent solution. Unbound primary antibody was then removed by washing in PBS. This was followed by incubation with fluorophore-conjugated secondary antibodies diluted in PBS, for 2 h at room temperature. The secondary antibodies were used at a dilution of 1:500 and were donkey anti-guinea-pig IgG Texas Red (Jackson, West Grove, PA, USA, Cat. 706-075-148) and goat anti-rabbit IgG fluorescein isothiocyanate (FITC) (Vector, Cat. FI-1000). Unbound antibody was removed with further PBS washing. Preparations were placed on glass microscope slides and coverslipped with Vectashield Mounting Medium for fluorescence (Vector, Cat. H-1000). Specimens were examined using a Zeiss Axioplan 2 Bio-Rad MRC 1024 confocal microscope and a conventional fluorescence microscope (Olympus BX61). Basic digital image processing was applied to images,
Single labelling Each opioid receptor antibody produced immunoreactive staining in the myenteric and submucous plexuses, with broadly consistent cellular distribution patterns between the plexuses. Immunoreactivity was not observed in muscle tissue. Strong, granular A-ir was present in the cytoplasm of several myenteric and submucous plexus cell bodies (Figs. 1A,D and 2A,D). However, A-ir was weak in projections, including interganglion fibre bundles and the tertiary plexus. A set of smaller (10 – 20 Am), ovoid, generally bipolar interstitial cells also showed weak to moderate cytoplasmic or membrane A-ir over the cell body. These cells were generally situated adjacent to, and aligned with myenteric plexus structures, in the same plane as the myenteric plexus (Fig. 1A,D). Interstitial A-ir cells of a similar size were also seen throughout the submucous plexus preparations, some of which were similarly located in close proximity to plexus structures (Fig. 2A). Intense, widespread yir was observed in both nerve plexuses (Figs. 1B and 2B). Staining appeared in the cytoplasm and on the surface of a large number of uni- and multipolar neurons in myenteric and submucous plexus ganglia. Staining extended to inter-ganglionic fibre bundles and tertiary plexus fibres. Moderate-
Fig. 2. Confocal micrographs showing the distribution of opioid receptor immunoreactivities in the submucous plexus of the rat ileum. A, D: As in myenteric ganglia, A-ir is mainly confined to neuronal cell bodies. B: y-ir is also present in ganglion cell bodies, but is also visible in projections, as indicated by an asterisk (*). A y-ir interstitial cell is indicated by an arrowhead. D: n-ir is punctate in the submucous plexus, and, as in the myenteric plexus, skirts the margin of ganglion cell bodies and runs along inter-ganglion projections. A n-ir projection is indicated by an asterisk (*). C, F: Overlaid images. Scale bar: 50 Am.
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intensity y-ir was seen extensively in interstitial cells as described above. Interstitial cell y-ir was variously seen in the cell body cytoplasm and on the cell membranes. n-ir was intense, highly punctate, and mainly observed in nerve fibres (Figs. 1E and 2E). Stained fibres were densely concentrated in myenteric and submucous plexus inter-ganglionic fibre bundles and broadly dispersed in the tertiary plexus. Staining skirted the margins of myenteric and submucous plexus ganglion cells, which we also interpreted as being immunoreactive fibres or terminals, although cell body membrane staining cannot be excluded. In the tertiary plexus, the pattern of n-ir most clearly suggested localisation in varicosities. n-ir was not visible within ganglion cell bodies. In submucous plexus preparations, some ovoid interstitial cells showed perikaryal n-ir. In submucous plexus preparations, strongly autofluorescent cells were noted in the vicinity of the crypts, possibly concealing some specific staining. These small cells were roughly the same size as the immunoreactive interstitial cells. These were particularly conspicuous when observing FITC fluorescence. Double labelling Cellular co-localisation of A and y immunoreactivity was extensive in ganglion cell bodies in both nerve plexuses. There were, however, some cells in the ganglia which appeared to express only one of the two types, i.e. A or y. Across the tissues obtained from three animals, 308 myenteric ganglia cell bodies were counted that were immunoreactive for A and/or y receptors. Of these, 228 (74%) showed co-localisation of A and y receptors in the cell body. 58 (19%) cell bodies were immunoreactive for A only, and the remaining 22 (7%) were immunoreactive for y only. These ratios approximate to those seen in each individual rat (rat 1, 104 cells counted, 68 (65%) A/y co-localised, 22 (21%) A only, 14 (13%) y only; rat 2, 109 cells counted, 81 (74%) A/y co-localised, 23 (21%) A only, 5 (5%) y only; rat 3, 95 cells counted, 79 (83%) A/y co-localised, 13 (14%) A only, 3 (3%) y only). Interstitial cells situated in close proximity to myenteric plexus structures typically showed A/y co-expression, although A-ir was often very weak. In the submucous plexus, all observed A-ir and y-ir ganglion cell bodies exhibited A/y co-localisation. A/y co-localisation was also frequently seen in interstitial cells in submucous plexus preparations, as was A/n co-localisation. Cellular co-expression of A and n receptors was impossible to verify in ganglia, due to the differing intracellular staining patterns seen. However, n-stained fibres were visibly apposed against A-ir cell bodies in ganglia of both plexuses (Figs. 1E,F and 2E,F). n fibres were also apposed against non-A-ir ganglion cells. Discussion The present study indicates that A-, y- and n-opioid receptors are broadly expressed across the neuronal myenteric and submucous plexus tissue in the rat ileum, with expression
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also occurring in interstitial cells. This is also the first immunohistochemical demonstration of y-opioid receptors in rat gastrointestinal tissue. Neurons of the enteric nervous system (ENS) form a network running the length of the gastrointestinal tract, and coordinate and regulate several gastrointestinal functions, including muscle motor and secretomotor activity, and nociception (for a review, see Hansen, 2003). The confocal images of A-, n- and y-opioid receptor-ir obtained in this study clearly showed neuronal and ICC-like cells. Further, secretomotor studies of ours and others have shown opioid receptors to be localised to nerve elements. It therefore seems credible that the ENS opioid receptors detected in this study are (at least in part) the sites of functional pharmacological action of opioids in the gut. As in other studies of this nature, identification of neurons was based on observation of cell morphologies. We have also observed extensive cellular co-localisation of A- and y-ir in myenteric ganglia with immunoreactivity to the neural marker, Hu (unpublished data). Comprehensive chemical coding of opioid receptor-expressing cells has not been performed in this study, but is expected to provide further insights into opioid mechanisms in the future. Bagnol et al. (1997) proposed that the opioid receptorexpressing interstitial cells in the rat small intestine might be interstitial cells of Cajal (ICC). ICCs are associated with various functions involved with motility regulation, including stretch reception, pacemaker signal generation, conduction of signalling between the ENS and the musculature, and amplification and propagation of inhibitory neurotransmission (Lecci et al., 2002). Double immunohistochemical labelling of opioid receptors with ICC markers, such as c-kit, may be useful in answering this question. Such a study in the guinea-pig ileum indicated that A-ir was not detected in interstitial cells (Ho et al., 2003). It is possible that some of these cells we observed belong to the deep muscular plexus, as observed by Ward and Sanders (2001), however the adjacent interstitial cells we saw were in the same plane as the ganglia and fibre bundles of the submucous plexus ganglia, and therefore it is unlikely that the deep muscular plexus interstitial cells account for all of the cells of this morphology that we observed in the submucous plexus tissue. In summary, the pattern of opioid receptor staining in interstitial cells seen in the present study does not appear to correspond with the pattern of c-kit distribution in the rat reported by Horiguchi and Komuro (1998) and Ward and Sanders (2001) who found the cells in both myenteric and deep muscular plexuses; clearly therefore further characterisation of the A-ir cells we observed is necessary. The A and n staining patterns are in general agreement with earlier reports of receptor expression (Bagnol et al., 1997; Fickel et al., 1997; Poonyachoti et al., 2001; Sternini et al., 1995). However, the absence of n-ir from ganglion cell bodies and interstitial cells near the myenteric plexus differs. It is possible that opioid receptor internalisation processes in enteric neurons (e.g., in response to endogenous opioids) could have contributed to these differences (McConalogue et al., 1999; Sternini et al., 1996). Gender, strain and handling and other
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stress variables in the animal could potentially be influential factors in such processes—for example female rats were used in the present study whilst male rats were used in the other studies referred to above. Secondly, previous studies have employed peroxidase staining methodologies, and the use of fluorescence staining may have introduced differences in detection sensitivity. Electron microscopy might provide for greater detection sensitivity, as well as spatial resolution in future studies. Thirdly, the anti-n antibody used in the present study was raised to a different epitope to those which have been used for previous reports. Immunohistochemical detection can be sensitive to the choice of epitope, but the reasons for this are often not clear. For example, apparently different intracellular localisations have been revealed by antibodies labelling C- and N-terminals of the y receptor in the CNS (Cahill et al., 2001). Splice variation of genes may play a role with this; isoforms of the A receptor gene are differentially localised in the CNS (MOR-1 is the major A isoform) (Abbadie et al., 2000). Splice variation is not currently well characterised for n or y receptors. The array of commercially available anti-opioid receptor antibodies has grown rapidly in recent years and now provides considerable scope for further investigation. One possibility would be to study receptor localization and coexpression by applying in situ hybridization in combination with immunohistochemistry or double in situ hybridization histochemistry. The extensive pattern of A/y co-expression in neurons and interstitial cells detected in this study is interesting, given that these receptors subserve identical effects on gastrointestinal transit and muscle contractility (Gray et al., 2005; De Winter et al., 1997; Hancock and Coupar, 1994; La Regina et al., 1988; Tavani et al., 1990). Furthermore, the relatively distinct distributions of A and n receptors fits with the disparate functional properties of these receptors in contractility and transit (Gray et al., 2005; La Regina et al., 1988; Tavani et al., 1990, De Winter et al., 1997). This overall pattern is in keeping with an established theme in opioid receptor pharmacology, whereby A and y receptors exhibit functional similarities, cooperativity, and synergism phenomena, and A and n receptors exhibit dissimilarity and even functional antagonism (Smith and Lee, 2003). Cellular A/y co-localisation has previously been shown in rat brain and spinal cord (Arvidsson et al., 1995; Cheng et al., 1997; Wang and Pickel, 2001) and mouse hypogastric ganglion neurons (in the absence of n receptors) (Rogers and Henderson, 1990). The electrophysiological effects of A and y receptor activation in guinea-pig enteric neurons appear identical, and are different to those of n receptor activation (Cherubini and North, 1985; Egan and North, 1981). In the CNS, A and y receptors typically interact to produce pharmacological synergism, whereas the relationship between A and n receptors is antagonistic (Pan, 1998; Schmidt et al., 2002). The present staining patterns suggest that A receptors are poised to act post-junctionally (and possibly pre-junctionally), y receptors post-junctionally and pre-junctionally, and n receptors pre-junctionally in neurons. The extent to which these intracellular localisations may be correlated with avail-
able functional data from the rat is limited, since appropriate functional data is quite scarce. The complete lack of positive functional n receptor data from the rat gastrointestinal tract makes drawing correlations particularly difficult, if not impossible. More extensive functional characterisations have been completed in the guinea-pig, and some findings appear to coincide with the present results (Johnson et al., 1988; Surprenant and North, 1985). However, profound functional differences in gastrointestinal opioid pharmacology have been demonstrated between the rat and guinea-pig, particularly with respect to n receptors (Gray et al., 2005). The epitope sequences to which the primary antibodies used in this study had been raised were unique to the intended targets and represented regions of high divergence between opioid receptor types. The labelling specificity of each antibody has previously been established with testing. The anti-A and anti-y antibodies have been used extensively in the immunohistochemical literature (Table 1). Anti-A antibodies raised to the same C-terminal epitope, but in rabbits, have also been tested for specificity and used widely in immunohistochemical characterisations (Arvidsson et al., 1995; McConalogue et al., 1999), and appear to produce equivalent results. We have found the anti-n antibody to produce expected staining patterns in the rat CNS (unpublished data). For example, the antibody produces intense staining in the superficial dorsal horn of the rat spinal cord and also in the substantia nigra, ventral tegmental area and hypothalamus. Conclusion The high rate of cellular A/y co-expression observed in this study indicates the potential for intracellular receptor interactions to occur, such as heterodimerisation and shared second messenger systems. The rat gastrointestinal tract may therefore provide a useful medium for the future study of these interactions and their significance in higher levels of tissue function. Acknowledgements The authors wish to thank Ms. Heather Robbins for her valuable technical support in this work. Andrew Gray is grateful for the support provided for this work by a Monash Graduate Scholarship. References Abbadie, C., Pan, Y.X., Pasternak, G.W., 2000. Differential distribution in rat brain of mu opioid receptor carboxy terminal splice variants MOR-1C-like and MOR-1-like immunoreactivity: evidence for region-specific processing. Journal of Comparative Neurology 419 (2), 244 – 256. Arvidsson, U., Riedl, M., Chakrabarti, S., Lee, J.H., Nakano, A.H., Dado, R.J., Loh, H.H., Law, P.Y., Wessendorf, M.W., Elde, R., 1995. Distribution and targeting of a mu-opioid receptor (MOR1) in brain and spinal cord. Journal of Neuroscience 15 (5 Pt 1), 3328 – 3341. Bagnol, D., Mansour, A., Akil, H., Watson, S.J., 1997. Cellular localization and distribution of the cloned mu and kappa opioid receptors in rat gastrointestinal tract. Neuroscience 81 (2), 579 – 591.
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La Regina, A., Petrillo, P., Sbacchi, M., Tavani, A., 1988. Interaction of U69,593 with mu-, alpha- and kappa-opioid binding sites and its analgesic and intestinal effects in rats. Life Sciences 42 (3), 293 – 301. Lecci, A., Santicioli, P., Maggi, C.A., 2002. Pharmacology of transmission to gastrointestinal muscle. Current Opinion in Pharmacology 2 (6), 630 – 641. McConalogue, K., Grady, E.F., Minnis, J., Balestra, B., Tonini, M., Brecha, N.C., Bunnett, N.W., Sternini, C., 1999. Activation and internalization of the mu-opioid receptor by the newly discovered endogenous agonists, endomorphin-1 and endomorphin-2. Neuroscience 90 (3), 1051 – 1059. Nishimura, E., Buchan, A.M., McIntosh, C.H., 1986. Autoradiographic localization of mu- and delta-type opioid receptors in the gastrointestinal tract of the rat and guinea pig. Gastroenterology 91 (5), 1084 – 1094. Pan, Z.Z., 1998. mu-Opposing actions of the kappa-opioid receptor. Trends in Pharmacological Sciences 19 (3), 94 – 98. Persson, P.A., Thorlin, T., Ronnback, L., Hansson, E., Eriksson, P.S., 2000. Differential expression of delta opioid receptors and mRNA in proliferating astrocytes during the cell cycle. Journal of Neuroscience Research 61 (4), 371 – 375. Pickel, V.M., Chan, J., Kash, T.L., Rodriguez, J.J., MacKie, K., 2004. Compartment-specific localization of cannabinoid 1 (CB1) and muopioid receptors in rat nucleus accumbens. Neuroscience 127 (1), 101 – 112. Poonyachoti, S., Portoghese, P.S., Brown, D.R., 2001. Characterization of opioid receptors modulating neurogenic contractions of circular muscle from porcine ileum and evidence that delta- and kappa-opioid receptors are coexpressed in myenteric neurons. Journal of Pharmacology and Experimental Therapeutics 297 (1), 69 – 77. Popper, P., Cristobal, R., Wackym, P.A., 2004. Expression and distribution of mu opioid receptors in the inner ear of the rat. Neuroscience 129 (1), 225 – 233. Porreca, F., Takemori, A.E., Sultana, M., Portoghese, P.S., Bowen, W.D., Mosberg, H.I., 1992. Modulation of mu-mediated antinociception in the mouse involves opioid delta-2 receptors. Journal of Pharmacology and Experimental Therapeutics 263 (1), 147 – 152. Rodriguez, J.J., Mackie, K., Pickel, V.M., 2001. Ultrastructural localization of the CB1 cannabinoid receptor in mu-opioid receptor patches of the rat caudate putamen nucleus. Journal of Neuroscience 21 (3), 823 – 833. Rogers, H., Henderson, G., 1990. Activation of mu- and delta-opioid receptors present on the same nerve terminals depresses transmitter release in the mouse hypogastric ganglion. British Journal of Pharmacology 101 (3), 505 – 512. Schmidt, B.L., Tambeli, C.H., Levine, J.D., Gear, R.W., 2002. mu/delta Cooperativity and opposing kappa-opioid effects in nucleus accumbensmediated antinociception in the rat. European Journal of Neuroscience 15 (5), 861 – 868. Sigma-Aldrich, 2004. Anti-opioid kappa receptor O1757 Product Information. Smith, A.P., Lee, N.M., 2003. Opioid receptor interactions: local and nonlocal, symmetric and asymmetric, physical and functional. Life Sciences 73 (15), 1873 – 1893. Sternini, C., Spann, M., De Giorgio, R., Anton, B., Keith Jr., D.E., Evans, C., Brecha, N.C., 1995. Cellular localization of the mu opioid receptor in the rat and guinea pig enteric nervous system. Analgesia 1 (4-6), 762 – 765. Sternini, C., Spann, M., Anton, B., Keith Jr., D.E., Bunnett, N.W., von Zastrow, M., Evans, C., Brecha, N.C., 1996. Agonist-selective endocytosis of mu opioid receptor by neurons in vivo. Proceedings of the National Academy of Sciences of the United States of America 93 (17), 9241 – 9246. Su, X., Joshi, S.K., Kardos, S., Gebhart, G.F., 2002. Sodium channel blocking actions of the kappa-opioid receptor agonist U50,488 contribute to its visceral antinociceptive effects. Journal of Neurophysiology 87 (3), 1271 – 1279. Surprenant, A., North, R.A., 1985. mu-Opioid receptors and alpha 2adrenoceptors coexist on myenteric but not on submucous neurones. Neuroscience 16 (2), 425 – 430. Tavani, A., Gambino, M.C., Petrillo, P., 1984. The opioid kappa-selective compound U-50,488H does not inhibit intestinal propulsion in rats. Journal of Pharmacy and Pharmacology 36 (5), 343 – 344.
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Tavani, A., Petrillo, P., La Regina, A., Sbacchi, M., 1990. Role of peripheral mu, delta and kappa opioid receptors in opioid-induced inhibition of gastrointestinal transit in rats. Journal of Pharmacology and Experimental Therapeutics 254 (1), 91 – 97. Varona, A., Gil, J., Saracibar, G., Maza, J.L., Echevarria, E., Irazusta, J., 2003. Effects of imipramine treatment on delta-opioid receptors of the rat brain cortex and striatum. Arzneimittel-Forschung 53 (1), 21 – 25. Wang, H., Pickel, V.M., 2001. Preferential cytoplasmic localization of deltaopioid receptors in rat striatal patches: comparison with plasmalemmal muopioid receptors. Journal of Neuroscience 21 (9), 3242 – 3250.
Ward, S.M., Sanders, K.M., 2001. Physiology and pathophysiology of the Interstitial Cell of Cajal: from bench to bedside: I. Functional development and plasticity of interstitial cells of Cajal networks. American Journal of Physiology: Gastrointestinal and Liver Physiology 281, G602 – G611. Xu, H., Partilla, J.S., de Costa, B.R., Rice, K.C., Rothman, R.B., 1993. Differential binding of opioid peptides and other drugs to two types of opioid delta ncx binding sites in mouse brain: further evidence for delta receptor heterogeneity. Peptides 14 (5), 893 – 907.
Comparison of opioid receptor distributions in the rat ileum A.C. Gray, I.M. Coupar, P.J. White * Department of Pharmaceutical Biology and Pharmacology, Victorian College of Pharmacy, Monash University, 381 Royal Pde, Parkville, Victoria 3052, Australia Received 17 April 2005; accepted 26 July 2005
Abstract The cellular expression patterns of A-, y- and n-opioid receptors in the rat ileum were examined using fluorescence immunohistochemistry. Double-labelling was used to examine cellular receptor co-localisation as a pre-requisite for intracellular molecular interactions, such as heterodimerisation. Tissues were stained as whole-mount preparations. Strong, broadly distributed immunoreactivity (ir) was observed for each receptor in the myenteric and submucous plexuses. Although intracellular A- and y-ir patterns differed in ganglion neurons, A/y co-expression was extensive in these cells. A/y co-expression was also observed in interstitial cells, which were diffusely distributed in submucous plexus preparations but generally located adjacent to myenteric plexus structures. Punctate n-ir was seen broadly in nerve fibres in both plexuses, suggesting localisation in varicosities. Neuronal A/n co-localisation was not apparent, although n-ir fibres were often apposed against A-ir cells. A/ n co-localisation was detected in interstitial cells in submucous plexus preparations. Similarities in A and y expression patterns might reflect similar functional properties previously detected for these receptors. This study indicates that the rat gastrointestinal tract might provide a useful tool for the future study of molecular interactions between opioid receptor types. D 2005 Elsevier Inc. All rights reserved. Keywords: y-opioid receptor; Interstitial cells; n-opioid receptor; A-opioid receptor; Myenteric plexus; Submucous plexus
Introduction Opioid drugs strongly inhibit gastrointestinal transit, which, depending on the clinical circumstances, can be either deleterious (as a side-effect of opioid analgesic treatment) or beneficial (in the treatment of diarrhoea). Biochemical transduction of these effects involves molecular binding of the drug to opioid receptors located within the gut wall. The three wellestablished opioid receptor types, A, y and n, have each been shown to carry responsibility for opioid gastrointestinal effects in studies across various species. All three types are present in the intestinal wall of the rat (Bagnol et al., 1997; Dashwood et al., 1985; Fickel et al., 1997; Gray et al., 2005; Nishimura et al., 1986; Sternini et al., 1995). A and y receptor activation each lead to gastrointestinal transit arrest in the rat (La Regina et al., 1988; Tavani et al., 1984, 1990), and our group has recently
Abbreviations: A; A-opioid receptor; y; y-opioid receptor; n; n-opioid receptor; DMSO; dimethyl sulfoxide; FIT; Cluorescein isothiocyanate; ICC; interstitial cells of Cajal; ir; Immunoreactive (-ity); PBS; phosphate buffered saline. * Corresponding author. Tel.: +61 3 9903 9074; fax: +61 3 9903 9638. E-mail address: [email protected] (P.J. White). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.07.048
shown that these receptors mediate inhibition of electrically induced neurogenic smooth muscle contractions in the rat ileum (Gray et al., 2005). A receptors also appear to mediate anti-secretory effects (Coupar, 1983). Despite investigations into gastrointestinal transit (Tavani et al., 1984), muscle contractility (Gray et al., 2005), secretion (Coupar, 1983) and visceral antinociception (Su et al., 2002), the functional effects of rat enteric n receptor activation remain unclear. Immunohistochemistry has shown that A and n receptors are localised in enteric neurons and interstitial cells in the rat gut (Bagnol et al., 1997; Fickel et al., 1997; Sternini et al., 1995). The identity of these interstitial cells is not currently known. y receptors have only been localised histochemically using autoradiography, at considerably lower resolution (Nishimura et al., 1986). However, functional pharmacological evidence suggests neuronal localisation (Hancock and Coupar, 1994). Opioid receptors appear to possess the ability to form dimers and/or larger oligomers, with the effect of altering various receptor properties (Jordan and Devi, 1999). Effects may include pharmacological synergism, ligand affinity changes, cross-tolerance and cross-dependence. Heterodimerised opioid receptors may be responsible for the existence of pharmacologically definable opioid receptor types in the
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Table 1 Primary antibodies used for staining Molecular target
Host
Epitope sequence
Source
Dilution
A-opioid receptor (MOR-1)
Guinea-pig
NHQLENLEAETAPLP (C-terminal)
y-opioid receptor (DOR-1)
Rabbit
LVPSARAELQSSPLV (N-terminal)
Chemicon (Temecula, CA, USA), Cat. AB1774 Chemicon, Cat. AB1560
n-opioid receptor (KOR-1)
Rabbit
Internal region
Sigma, Cat. O1757
absence of single, isolatable corresponding genetic entities. An example is the y2 receptor, which is putatively dimerised A and y receptors (Gomes et al., 2000; Porreca et al., 1992; Xu et al., 1993). Better understanding of receptor oligomerisation may provide benefits to clinical drug use and drug development in the future. A logical requirement for oligomerisation is cellular co-localisation of the receptor proteins. Other potential intracellular interactions could occur at second messenger or subsequent levels, and would also necessitate co-localisation (Smith and Lee, 2003). It has been speculated that A and n receptors might be co-expressed by rat enteric neurons, since these receptors exhibit similar anatomical distributions, as revealed by immunohistochemistry (Fickel et al., 1997). Coexpression of A and n, and A and y receptors has already been demonstrated in guinea-pig ileum neurons using electrophysiological methods (Cherubini and North, 1985; Egan and North, 1981). Double-label immunohistochemistry has been used to show neuronal co-expression of y and n receptors in the pig ileum (Poonyachoti et al., 2001). The aim of this study was to establish whether the potential exists for intracellular and cellular opioid receptor interactions in the rat small intestine. Immunohistochemistry and doublelabel immunohistochemistry were used. The distributions of A,
1:200 – 1:500
1:500 – 1:1000
1:200 – 1:500
References (Pickel et al., 2004; Popper et al., 2004; Rodriguez et al., 2001) (Cahill et al., 2001; Commons and Milner, 1996, 1997; Persson et al., 2000; Varona et al., 2003) (Sigma-Aldrich, 2004)
y and n opioid receptors were examined, as was the extent to which cells express y and n receptors together with A receptors. Part of this study was presented at the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists (ASCEPT) meeting in 2003 (Gray et al., 2003). Materials and methods Animals and tissue preparation The tissue preparation method was similar to that of Costa et al. (1980). Three female Hooded Wistar rats (250 –300 g) (Victorian College of Pharmacy, Monash University, Australia) were heavily anaesthetised with thiopentone (120 mg/kg, i.p.), and transcardially perfused with 200 ml phosphate buffered saline, pH 7.4 (PBS), followed by 200 ml 4% formaldehyde in PBS. Terminal ileum segments (10 cm proximal to the caecum) were excised and thoroughly rinsed with PBS. The segments were sectioned along the mesenteric border and pinned flat and taut on balsawood boards (with the mucosa apposed), and postfixed in 4% formaldehyde in PBS overnight at 4 -C. The tissues were cleared with three 10 min washes in DMSO (as per the protocol of Grace and Llinas, 1985), which were followed
Fig. 1. Confocal micrographs showing the distribution of opioid receptor immunoreactivities in the myenteric plexus of the rat ileum. A, D: A-ir is visible in the cytoplasm of neuronal cell bodies in ganglia, but is weak or absent in fibres. Some lightly stained interstitial cells are located adjacent to plexus structures (indicated by arrowheads). An asterisk (*) indicates a neuron that has stained positive for A receptors, but not y receptors. B: y-ir extends to the tertiary plexus and is colocalised with A receptors in ganglia and interstitial cells. E: Punctate n-ir skirts ganglion cell bodies and extends into the tertiary plexus. A n-ir varicose fibre passes against a lightly-stained A-ir interstitial cell (indicated by an arrowhead) in the tertiary plexus. C, F: Overlaid images. Scale bar: 50 Am.
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by another set of PBS washes. The ileum wall layers were separated by manual microdissection to produce separate whole-mount preparations, enabling en face examination of the myenteric and submucous plexuses and adherent smooth muscle. Ethical approval was obtained from the Monash University Victorian College of Pharmacy Animal Experimentation Ethics Committee. All efforts were made to minimise the suffering and the number of animals used.
involving adjustments to contrast, brightness and gamma. Stained cells were counted manually to quantify the prevalence of receptor co-localisation. Staining controls, involving omission of either or both primary antibodies, were routinely prepared and imaged to establish specificity of staining. Levels of autofluorescence and non-specific secondary antibody binding were generally found to be very low.
Immunohistochemistry
Results
Preparations were incubated for 30 min in an antibody diluent solution, which contained 0.3% Triton X-100 (Sigma, St. Louis, MO, USA), 3% normal goat serum (Vector, Burlingame, CA, USA), 1% bovine serum albumin (Sigma) and 0.01% sodium azide (Sigma) in PBS. Following this, mixtures of primary antibodies labelling A and y, or A and n receptors (Table 1), were incubated with the free-floating preparations for 24 –48 h at 4 -C, in the same antibody diluent solution. Unbound primary antibody was then removed by washing in PBS. This was followed by incubation with fluorophore-conjugated secondary antibodies diluted in PBS, for 2 h at room temperature. The secondary antibodies were used at a dilution of 1:500 and were donkey anti-guinea-pig IgG Texas Red (Jackson, West Grove, PA, USA, Cat. 706-075-148) and goat anti-rabbit IgG fluorescein isothiocyanate (FITC) (Vector, Cat. FI-1000). Unbound antibody was removed with further PBS washing. Preparations were placed on glass microscope slides and coverslipped with Vectashield Mounting Medium for fluorescence (Vector, Cat. H-1000). Specimens were examined using a Zeiss Axioplan 2 Bio-Rad MRC 1024 confocal microscope and a conventional fluorescence microscope (Olympus BX61). Basic digital image processing was applied to images,
Single labelling Each opioid receptor antibody produced immunoreactive staining in the myenteric and submucous plexuses, with broadly consistent cellular distribution patterns between the plexuses. Immunoreactivity was not observed in muscle tissue. Strong, granular A-ir was present in the cytoplasm of several myenteric and submucous plexus cell bodies (Figs. 1A,D and 2A,D). However, A-ir was weak in projections, including interganglion fibre bundles and the tertiary plexus. A set of smaller (10 – 20 Am), ovoid, generally bipolar interstitial cells also showed weak to moderate cytoplasmic or membrane A-ir over the cell body. These cells were generally situated adjacent to, and aligned with myenteric plexus structures, in the same plane as the myenteric plexus (Fig. 1A,D). Interstitial A-ir cells of a similar size were also seen throughout the submucous plexus preparations, some of which were similarly located in close proximity to plexus structures (Fig. 2A). Intense, widespread yir was observed in both nerve plexuses (Figs. 1B and 2B). Staining appeared in the cytoplasm and on the surface of a large number of uni- and multipolar neurons in myenteric and submucous plexus ganglia. Staining extended to inter-ganglionic fibre bundles and tertiary plexus fibres. Moderate-
Fig. 2. Confocal micrographs showing the distribution of opioid receptor immunoreactivities in the submucous plexus of the rat ileum. A, D: As in myenteric ganglia, A-ir is mainly confined to neuronal cell bodies. B: y-ir is also present in ganglion cell bodies, but is also visible in projections, as indicated by an asterisk (*). A y-ir interstitial cell is indicated by an arrowhead. D: n-ir is punctate in the submucous plexus, and, as in the myenteric plexus, skirts the margin of ganglion cell bodies and runs along inter-ganglion projections. A n-ir projection is indicated by an asterisk (*). C, F: Overlaid images. Scale bar: 50 Am.
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intensity y-ir was seen extensively in interstitial cells as described above. Interstitial cell y-ir was variously seen in the cell body cytoplasm and on the cell membranes. n-ir was intense, highly punctate, and mainly observed in nerve fibres (Figs. 1E and 2E). Stained fibres were densely concentrated in myenteric and submucous plexus inter-ganglionic fibre bundles and broadly dispersed in the tertiary plexus. Staining skirted the margins of myenteric and submucous plexus ganglion cells, which we also interpreted as being immunoreactive fibres or terminals, although cell body membrane staining cannot be excluded. In the tertiary plexus, the pattern of n-ir most clearly suggested localisation in varicosities. n-ir was not visible within ganglion cell bodies. In submucous plexus preparations, some ovoid interstitial cells showed perikaryal n-ir. In submucous plexus preparations, strongly autofluorescent cells were noted in the vicinity of the crypts, possibly concealing some specific staining. These small cells were roughly the same size as the immunoreactive interstitial cells. These were particularly conspicuous when observing FITC fluorescence. Double labelling Cellular co-localisation of A and y immunoreactivity was extensive in ganglion cell bodies in both nerve plexuses. There were, however, some cells in the ganglia which appeared to express only one of the two types, i.e. A or y. Across the tissues obtained from three animals, 308 myenteric ganglia cell bodies were counted that were immunoreactive for A and/or y receptors. Of these, 228 (74%) showed co-localisation of A and y receptors in the cell body. 58 (19%) cell bodies were immunoreactive for A only, and the remaining 22 (7%) were immunoreactive for y only. These ratios approximate to those seen in each individual rat (rat 1, 104 cells counted, 68 (65%) A/y co-localised, 22 (21%) A only, 14 (13%) y only; rat 2, 109 cells counted, 81 (74%) A/y co-localised, 23 (21%) A only, 5 (5%) y only; rat 3, 95 cells counted, 79 (83%) A/y co-localised, 13 (14%) A only, 3 (3%) y only). Interstitial cells situated in close proximity to myenteric plexus structures typically showed A/y co-expression, although A-ir was often very weak. In the submucous plexus, all observed A-ir and y-ir ganglion cell bodies exhibited A/y co-localisation. A/y co-localisation was also frequently seen in interstitial cells in submucous plexus preparations, as was A/n co-localisation. Cellular co-expression of A and n receptors was impossible to verify in ganglia, due to the differing intracellular staining patterns seen. However, n-stained fibres were visibly apposed against A-ir cell bodies in ganglia of both plexuses (Figs. 1E,F and 2E,F). n fibres were also apposed against non-A-ir ganglion cells. Discussion The present study indicates that A-, y- and n-opioid receptors are broadly expressed across the neuronal myenteric and submucous plexus tissue in the rat ileum, with expression
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also occurring in interstitial cells. This is also the first immunohistochemical demonstration of y-opioid receptors in rat gastrointestinal tissue. Neurons of the enteric nervous system (ENS) form a network running the length of the gastrointestinal tract, and coordinate and regulate several gastrointestinal functions, including muscle motor and secretomotor activity, and nociception (for a review, see Hansen, 2003). The confocal images of A-, n- and y-opioid receptor-ir obtained in this study clearly showed neuronal and ICC-like cells. Further, secretomotor studies of ours and others have shown opioid receptors to be localised to nerve elements. It therefore seems credible that the ENS opioid receptors detected in this study are (at least in part) the sites of functional pharmacological action of opioids in the gut. As in other studies of this nature, identification of neurons was based on observation of cell morphologies. We have also observed extensive cellular co-localisation of A- and y-ir in myenteric ganglia with immunoreactivity to the neural marker, Hu (unpublished data). Comprehensive chemical coding of opioid receptor-expressing cells has not been performed in this study, but is expected to provide further insights into opioid mechanisms in the future. Bagnol et al. (1997) proposed that the opioid receptorexpressing interstitial cells in the rat small intestine might be interstitial cells of Cajal (ICC). ICCs are associated with various functions involved with motility regulation, including stretch reception, pacemaker signal generation, conduction of signalling between the ENS and the musculature, and amplification and propagation of inhibitory neurotransmission (Lecci et al., 2002). Double immunohistochemical labelling of opioid receptors with ICC markers, such as c-kit, may be useful in answering this question. Such a study in the guinea-pig ileum indicated that A-ir was not detected in interstitial cells (Ho et al., 2003). It is possible that some of these cells we observed belong to the deep muscular plexus, as observed by Ward and Sanders (2001), however the adjacent interstitial cells we saw were in the same plane as the ganglia and fibre bundles of the submucous plexus ganglia, and therefore it is unlikely that the deep muscular plexus interstitial cells account for all of the cells of this morphology that we observed in the submucous plexus tissue. In summary, the pattern of opioid receptor staining in interstitial cells seen in the present study does not appear to correspond with the pattern of c-kit distribution in the rat reported by Horiguchi and Komuro (1998) and Ward and Sanders (2001) who found the cells in both myenteric and deep muscular plexuses; clearly therefore further characterisation of the A-ir cells we observed is necessary. The A and n staining patterns are in general agreement with earlier reports of receptor expression (Bagnol et al., 1997; Fickel et al., 1997; Poonyachoti et al., 2001; Sternini et al., 1995). However, the absence of n-ir from ganglion cell bodies and interstitial cells near the myenteric plexus differs. It is possible that opioid receptor internalisation processes in enteric neurons (e.g., in response to endogenous opioids) could have contributed to these differences (McConalogue et al., 1999; Sternini et al., 1996). Gender, strain and handling and other
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stress variables in the animal could potentially be influential factors in such processes—for example female rats were used in the present study whilst male rats were used in the other studies referred to above. Secondly, previous studies have employed peroxidase staining methodologies, and the use of fluorescence staining may have introduced differences in detection sensitivity. Electron microscopy might provide for greater detection sensitivity, as well as spatial resolution in future studies. Thirdly, the anti-n antibody used in the present study was raised to a different epitope to those which have been used for previous reports. Immunohistochemical detection can be sensitive to the choice of epitope, but the reasons for this are often not clear. For example, apparently different intracellular localisations have been revealed by antibodies labelling C- and N-terminals of the y receptor in the CNS (Cahill et al., 2001). Splice variation of genes may play a role with this; isoforms of the A receptor gene are differentially localised in the CNS (MOR-1 is the major A isoform) (Abbadie et al., 2000). Splice variation is not currently well characterised for n or y receptors. The array of commercially available anti-opioid receptor antibodies has grown rapidly in recent years and now provides considerable scope for further investigation. One possibility would be to study receptor localization and coexpression by applying in situ hybridization in combination with immunohistochemistry or double in situ hybridization histochemistry. The extensive pattern of A/y co-expression in neurons and interstitial cells detected in this study is interesting, given that these receptors subserve identical effects on gastrointestinal transit and muscle contractility (Gray et al., 2005; De Winter et al., 1997; Hancock and Coupar, 1994; La Regina et al., 1988; Tavani et al., 1990). Furthermore, the relatively distinct distributions of A and n receptors fits with the disparate functional properties of these receptors in contractility and transit (Gray et al., 2005; La Regina et al., 1988; Tavani et al., 1990, De Winter et al., 1997). This overall pattern is in keeping with an established theme in opioid receptor pharmacology, whereby A and y receptors exhibit functional similarities, cooperativity, and synergism phenomena, and A and n receptors exhibit dissimilarity and even functional antagonism (Smith and Lee, 2003). Cellular A/y co-localisation has previously been shown in rat brain and spinal cord (Arvidsson et al., 1995; Cheng et al., 1997; Wang and Pickel, 2001) and mouse hypogastric ganglion neurons (in the absence of n receptors) (Rogers and Henderson, 1990). The electrophysiological effects of A and y receptor activation in guinea-pig enteric neurons appear identical, and are different to those of n receptor activation (Cherubini and North, 1985; Egan and North, 1981). In the CNS, A and y receptors typically interact to produce pharmacological synergism, whereas the relationship between A and n receptors is antagonistic (Pan, 1998; Schmidt et al., 2002). The present staining patterns suggest that A receptors are poised to act post-junctionally (and possibly pre-junctionally), y receptors post-junctionally and pre-junctionally, and n receptors pre-junctionally in neurons. The extent to which these intracellular localisations may be correlated with avail-
able functional data from the rat is limited, since appropriate functional data is quite scarce. The complete lack of positive functional n receptor data from the rat gastrointestinal tract makes drawing correlations particularly difficult, if not impossible. More extensive functional characterisations have been completed in the guinea-pig, and some findings appear to coincide with the present results (Johnson et al., 1988; Surprenant and North, 1985). However, profound functional differences in gastrointestinal opioid pharmacology have been demonstrated between the rat and guinea-pig, particularly with respect to n receptors (Gray et al., 2005). The epitope sequences to which the primary antibodies used in this study had been raised were unique to the intended targets and represented regions of high divergence between opioid receptor types. The labelling specificity of each antibody has previously been established with testing. The anti-A and anti-y antibodies have been used extensively in the immunohistochemical literature (Table 1). Anti-A antibodies raised to the same C-terminal epitope, but in rabbits, have also been tested for specificity and used widely in immunohistochemical characterisations (Arvidsson et al., 1995; McConalogue et al., 1999), and appear to produce equivalent results. We have found the anti-n antibody to produce expected staining patterns in the rat CNS (unpublished data). For example, the antibody produces intense staining in the superficial dorsal horn of the rat spinal cord and also in the substantia nigra, ventral tegmental area and hypothalamus. Conclusion The high rate of cellular A/y co-expression observed in this study indicates the potential for intracellular receptor interactions to occur, such as heterodimerisation and shared second messenger systems. The rat gastrointestinal tract may therefore provide a useful medium for the future study of these interactions and their significance in higher levels of tissue function. Acknowledgements The authors wish to thank Ms. Heather Robbins for her valuable technical support in this work. Andrew Gray is grateful for the support provided for this work by a Monash Graduate Scholarship. References Abbadie, C., Pan, Y.X., Pasternak, G.W., 2000. Differential distribution in rat brain of mu opioid receptor carboxy terminal splice variants MOR-1C-like and MOR-1-like immunoreactivity: evidence for region-specific processing. Journal of Comparative Neurology 419 (2), 244 – 256. Arvidsson, U., Riedl, M., Chakrabarti, S., Lee, J.H., Nakano, A.H., Dado, R.J., Loh, H.H., Law, P.Y., Wessendorf, M.W., Elde, R., 1995. Distribution and targeting of a mu-opioid receptor (MOR1) in brain and spinal cord. Journal of Neuroscience 15 (5 Pt 1), 3328 – 3341. Bagnol, D., Mansour, A., Akil, H., Watson, S.J., 1997. Cellular localization and distribution of the cloned mu and kappa opioid receptors in rat gastrointestinal tract. Neuroscience 81 (2), 579 – 591.
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