Opioid, cannabinoid and vanilloid receptor localization on porcine cultured myenteric neurons Anjali Kulkarni-Narla, David R. Brown * Department of Veterinary PathoBiology, University of Minnesota, 1988 Fitch Avenue, St. Paul, MN 55108-6010, USA Received 7 March 2001; received in revised form 30 May 2001; accepted 4 June 2001 Abstract Opioids and cannabinoids have profound inhibitory actions on intestinal motility which are mediated in part by their cognate receptors in the enteric nervous system. In the present study, we examined the expression of immunoreactivity for d- and k-opioid receptors, CB 1 -cannabinoid receptors and type 1 vanilloid receptors by immunocytochemistry and confocal laser scanning microscopy on ileal myenteric neurons, isolated from juvenile pigs, that were , 70 mm diameter in either axis and maintained for 1–2 weeks in primary culture. Immunoreactivities for d-opioid and cannabinoid recep- tors were present in neurons immunoreactive for the cholinergic marker, choline acetyltransferase. Some neurons with d-opioid receptor-like immunoreactivity were also immunoreactive for k-opioid, cannabinoid or vanilloid receptors. These observations indicate that receptors for cannabinoids or vanilloids are co-localized in opioid receptor-expressing myenteric neurons which modulate intestinal sensorimotor function. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Gut; Enteric nervous system; Myenteric plexus; Receptors; Pig The enteric nervous system regulates patterns of motility in the intestine, including the regional mixing and propul- sion of gut contents, and propagated contractions associated with the migrating myoelectric complex [12]. The ability of opioids, such as codeine and loperamide, to inhibit intestinal propulsion and slow intestinal transit is a prominent compo- nent of the antidiarrheal and constipating actions of this drug class. Presynaptic opioid receptors (ORs) on intrinsic neurons in the myenteric plexus mediate opioid-induced neuronal hyperpolarization and reductions in the release of acetylcholine and other neurotransmitters through the G protein-coupled activation of K 1 channels or inhibition of N-type Ca 21 channel gating [7]. Like the opioids, botanical, endogenous and synthetic cannabinoids have been shown to inhibit neurotransmitter release and decrease intestinal motility through interactions with presynaptic CB 1 -cannabi- noid receptors (CB 1 -R) in the myenteric plexus [4,5,10]. In the porcine small intestine, a biomedical model for the human intestine, opioid agonists acting selectively at d- ORs (DOR) or k-ORs (KOR) decrease neurogenic contrac- tions of smooth muscle-myenteric plexus strips from porcine ileum [13]. In addition to these ORs, the porcine myenteric plexus exhibits relatively high levels of expres- sion for their cognate ligands, the enkephalins [14]. In support of these data, DOR and KOR immunoreactivities are expressed in myenteric neurons and fibers examined in tissue sections; in some neurons, these receptors appear to be colocalized [3,13]. In addition, CB 1 -R immunoreactivity has been found to be expressed in neurons within myenteric ganglia of the porcine ileum and colon that are also immu- noreactive for the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT) [11]. The availability of myenteric neurons in primary culture would enhance investigations of the biology and potential interactions between enteric opioid and cannabinoid recep- tors at the cellular and molecular level. In the present study, we developed a method for maintaining myenteric neurons from juvenile pigs in primary culture to test the hypothesis that under culture conditions, these neurons would manifest the opioid and cannabinoid receptor signatures observed in our previous immunohistochemical experiments in porcine small intestine [3,11,13]. As DOR immunoreactivity has been found in primary afferent fibers in dorsal horn of the spinal cord [6], it was of additional interest to examine the possible colocalization of these receptors on myenteric neurons with the type 1 vanilloid receptor (VR1), a Neuroscience Letters 308 (2001) 153–156 0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0304-3940(01)01998-X www.elsevier.com/locate/neulet * Corresponding author. Tel.: 11-612-624-0713; fax: 11-612- 625-0204. E-mail address: brown013@umn.edu (D.R. Brown).