Cannabis has been used for centuries in the medicinal treatment of gastrointestinal disorders. Endogenous cannabinimimetic substances such as 2-arachidonylglycerol have been isolated from gut homogenates and CB1-cannabinoid binding sites have been identified in small intestine. In this study, CB1-cannabinoid receptors (CB1-R) were immunohistochemically localized within the enteric nervous system of the pig, an omnivorous species whose digestive tract is functionally similar to humans. Two anti-CB1-R antisera, raised against N-terminal epitopes in the human CB1-R, were employed to localize receptor immunoreactivity by secondary immunofluorescence. CB1-R immunoreactivity was observed in the myenteric and submucosal ganglionated plexuses of porcine ileum and colon. In the ileum, all CB1-R-immunoreactive neurons coexpressed immunoreactivity to the cholinergic marker, choline acetyltransferase (ChAT). CB1-R/ChAT-immunoreactive neurons appeared to be in close apposition to ileal Peyer's patches, submucosal blood vessels, and intestinal crypts. In the distal colon, CB1-R-immunoreactive neurons also expressed immunoreactivity to ChAT, albeit less frequently than in ileum. Immunoreactivity to vasoactive intestinal peptide or nitric oxide synthase was not colocalized in ileal or colonic CB1-R-immunoreactive neurons. These studies indicate that CB1-R are present in cholinergic neurons in the porcine enteric nervous system. The potential roles of these receptors in intestinal motility and epithelial transport, host defense and visceral pain transmission are discussed.

1 The role of cannabinoid (CB) receptors in the regulation of gastric acid secretion was investigated in the rat by means of functional experiments and by immunohistochemistry. 2 In anaesthetized rats with lumen-perfused stomach, the non selective CB-receptor agonist WIN 55,212-2 (0.30 ± 4.00 mmol kg 71 , i.v.) and the selective CB 1 -receptor agonist HU-210 (0.03 ± 1.50 mmol kg 71 , i.v.), dose-dependently decreased the acid secretion induced by both pentagastrin (30 nmol kg 71 h 71 ) and 2-deoxy-D-glucose (1.25 mmol kg 71 , i.v.). By contrast, neither WIN 55,212-2 (1 ± 4 mmol kg 71 , i.v.) nor HU-210 (0.03 ± 1.50 mmol kg 71 , i.v.) did modify histamine-induced acid secretion (20 mmol kg 71 h 71 ). The selective CB 2 -receptor agonist JWH-015 (3 ± 10 mmol kg 71 , i.v.) was ine ective. 3 The gastric antisecretory e ects of WIN 55,212-2 and HU-210 on pentagastrin-induced acid secretion were prevented by the selective CB 1 -receptor antagonist SR141716A (0.65 mmol kg 71 , i.v.) and una ected by the selective CB 2 -receptor antagonist SR144528 (0.65 ± 2 mmol kg 71 , i.v.). 4 Bilateral cervical vagotomy and ganglionic blockade with hexamethonium (10 mg kg 71 , i.v., followed by continuous infusion of 10 mg kg 71 h 71 ) signi®cantly reduced, but not abolished, the maximal inhibitory e ect of HU-210 (0.3 mmol kg 71 , i.v.) on pentagastrin-induced acid secretion; by contrast, pretreatment with atropine (1 mg kg 71 , i.v.) did not modify the antisecretory e ect of HU-210. 5 Immunoreactivity to the CB 1 receptor was co-localized with that of the cholinergic marker choline acetyltransferase in neural elements innervating smooth muscle, mucosa and submucosal blood vessels of rat stomach fundus, corpus and antrum. In contrast, CB 2 receptor-like immunoreactivity was not observed. 6 These results indicate that gastric antisecretory e ects of cannabinoids in the rat are mediated by suppression of vagal drive to the stomach through activation of CB 1 receptors, located on pre-and postganglionic cholinergic pathways. However, the ine ectiveness of atropine in reducing the e ect of HU-210 suggests that the release of non cholinergic excitatory neurotransmitters may be regulated by CB 1 receptors.

With its abundance of neurons and immunocytes, the gut is a potentially important site for the study of the interaction between the nervous and immune systems. Using immunohistochemical techniques, we tested the hypothesis that gut-associated lymphoid tissue in the porcine small intestine might receive catecholaminergic, cholinergic and peptidergic innervation. Antibodies against protein gene product (PGP) 9.5 were employed to detect neuronal membranes; antibodies against tyrosine hydroxylase (TH), type 2 vesicular monoamine transporter (VMAT-2) and choline acetyltransferase (ChAT) were used to detect catecholaminergic and cholinergic neurons; and antibodies to neuromedin U-8 (NMU-8), substance P (SP) and vasoactive intestinal peptide (VIP) were also used. PGP9.5-immunoreactive nerve fibers were observed between jejunal Peyer's patch (PP) follicles and in submucosal ganglia localized at the base of continuous ileal PP. Many ChAT-positive and a few TH-/VMAT-2-immunoreactive neurons or axons adjacent to jejunal and ileal PP were observed. Neurons and fibers from ganglia situated between or at the base of PP follicles manifested robust immunoreactivities to VIP and NMU-8; relatively less SP immunoreactivity was observed at these locations. All neuromedin-U 8-positive neurons observed exhibited immunoreactivity to ChAT as did some VIP-positive neurons. The specific chemical coding of enteric neurons in close apposition to jejunal and ileal PP and the differential localization of neuropeptides within the jejunal and ileal PP are indicative of neuroimmunomodulation at these sites.

No abstract

Opioid drugs have profound antidiarrheal and constipating actions in the intestinal tract and are effective in mitigating abdominal pain. Mediators of intestinal inflammation and allergy produce increased mucosal secretion, altered bowel motility and pain due to their ability to evoke enteric secretomotor reflexes through primary afferent neurons. In this study, the distribution of delta- and kappa-opioid receptor (DOR and KOR, respectively) immunoreactivities in chemically identified neurons of the porcine ileum was compared with that of the capsaicin-sensitive type 1 vanilloid receptor (VR1). DOR and VR1 immunoreactivities were observed to be highly localized in choline acetyltransferase (ChAT)- and calcitonin gene-related peptide (CGRP)-positive neurons and nerve fibers of the submucosal and myenteric plexuses and both receptors exhibited frequent colocalization. In the inner submucosal plexus, they also were colocalized in substance P (SP)-positive neurons. Neurons in the outer submucosal plexus expressed DOR immunoreactivity alone or in combination with VR1. KOR-immunoreactive neurons were found only in the myenteric plexus; these cells coexpressed immunoreactivity to ChAT, CGRP, vasoactive intestinal peptide (VIP) or nitric oxide synthase (NOS). In addition, some KOR-positive neurons coexpressed immunoreactivities to DOR and VR1. Based on their neurochemical coding, opioid and vanilloid receptor-immunoreactive neurons in the submucosal and myenteric plexuses may include primary afferents and constitute novel therapeutic targets for the palliation of painful intestinal inflammatory, hypersensitivity and dysmotility states.

Enteric neurotransmitters can modulate the biodefensive functions of the intestinal mucosa, but their role in mucosal interactions with enteropathogens is not well defined. Here we tested the hypothesis that norepinephrine (NE) modulates interactions between enterohemorrhagic Escherichia coli O157:H7 (EHEC) and the colonic epithelium. Mucosal sheets from porcine distal colon were mounted in Ussing chambers. Drugs and an inoculum of either Shiga toxin-negative or -positive EHEC were added to the contraluminal and luminal bathing medium, respectively. After 90 min, adherent bacteria were quantified by an adherence assay and by immunohistochemical methods; short-circuit current (I(sc)) was measured continuously to assess changes in active ion transport. NE-treated tissues exhibited concentration-dependent increases in I(sc) and EHEC adherence. NE did not alter adherence of a rodent-adapted, noninfectious E. coli strain or two porcine-adapted non-O157 E. coli strains. The actions of NE on EHEC adherence but not I(sc) were prevented by the alpha-adrenergic antagonist yohimbine and the PKA activator Sp-8-bromoadenosine-3',5'-cyclic monophosphorothioate. Like NE, the PKA inhibitor Rp-8-bromoadenosine-3',5'-cyclic monophosphorothioate or indirectly acting sympathomimetic agents increased EHEC adherence. Nerve fibers immunoreactive for the NE-synthesizing enzymes tyrosine hydroxylase and dopamine beta-hydroxylase appeared to innervate the colonic epithelium. EHEC-like immunoreactivity on the colonic surface had the appearance of bacterial microcolonies and increased after NE treatment by a phentolamine-sensitive mechanism. Through interactions with alpha(2)-adrenergic receptors, NE appears to increase EHEC adherence to the colonic mucosa. Changes in sympathetic neural outflow may alter intestinal susceptibility to infection.

Impaired development of local Ab responses may predispose HIV-1-infected patients to an increased rate, severity, and duration of mucosal infections. We characterized the repertoire of Ig-producing cells in the intestinal effector compartment (the lamina propria) of HIV-1-infected (n = 29) and seronegative control (n = 27) subjects. The density of Ig-producing cells per area was similar in both groups. However, the proportions of IgA-producing cells were lower in both the duodenum and colon from HIV-1-infected patients compared with those of control subjects (p < 0.05), with compensatory increases in IgG-producing cells in the colon and IgM-producing cells in the duodenum. Similarly, among Abs in the lumen the proportions of IgA were also decreased and the proportions of IgG were increased among HIV-1-infected patients. On a molecular level, VH gene repertoire analyses by RT-PCR revealed comparable proportions of the VH3 family among duodenal IgA transcripts (50–53%) from both groups. VH3 expression was decreased only for IgM among patients with advanced HIV-1 disease (n = 6) compared with that of control subjects (n = 8) (48 ± 8 vs 62 ± 13%; p < 0.01). Moreover, the frequencies of individual IgM and IgA VH3 genes were comparable in each group, including rates of putative HIV-1 gp120-binding VH3 genes (V3-23, V3-30, V3-30/3-30.5). We conclude that, despite a decrement in local IgA producing cells, the density and molecular VH repertoire of mucosal plasma cells are relatively intact among patients with HIV-1 infection. These data suggest that HIV-1-infected patients use functional regulatory mechanisms to provide sufficient VH diversity and effective induction and differentiation of mucosal B cells.

No abstract