Winnie Ho

Effects of cannabinoid receptor-2 activation on accelerated gastrointestinal transit in lipopolysaccharide-treated rats.

The biological effects of cannabinoids (CB) are mediated by CB1 and CB2 receptors. The role of CB2 receptors in the gastrointestinal tract is uncertain. In this study, we examined whether CB2 receptor activation is involved in the regulation of gastrointestinal transit in rats. Basal and lipopolysaccharide (LPS)‐stimulated gastrointestinal transit was measured after instillation of an Evans blue‐gum Arabic suspension into the stomach, in the presence of specific CB1 and CB2 agonists and antagonists, or after treatment with inhibitors of mediators implicated in the transit process. In control rats a CB1 (ACEA; 1 mg kg−1), but not a CB2 (JWH‐133; 1 mg kg−1), receptor agonist inhibited basal gastrointestinal transit. The effects of the CB1 agonist were reversed by the CB1 antagonist AM‐251, which alone increased basal transit. LPS treatment increased gastrointestinal transit. This increased transit was reduced to control values by the CB2, but not the CB1, agonist. This inhibition by the CB2 agonist was dose dependent and prevented by a selective CB2 antagonist (AM‐630; 1 mg kg−1). By evaluating the inhibition of LPS‐enhanced gastrointestinal transit by different antagonists, the effects of the CB2 agonist (JWH‐133; 1 mg kg−1) were found to act via cyclooxygenase, and to act independently of inducible nitric oxide synthase (NOS) and platelet‐activating factor. Interleukin‐1β and constitutive NOS isoforms may be involved in the accelerated LPS transit. The activation of CB2 receptors in response to LPS is a mechanism for the re‐establishment of normal gastrointestinal transit after an inflammatory stimulus.

Cyclooxygenase-2 contributes to dysmotility and enhanced excitability of myenteric AH neurones in the inflamed guinea pig distal colon.

We have previously demonstrated that trinitrobenzene sulphonic acid (TNBS)‐induced colitis in guinea pig is associated with hyperexcitability of myenteric AH neurones, enhanced synaptic activity in the myenteric plexus, increased serotonin (5‐HT) availability in the mucosa, and decreased propulsive motor activity. The current study tested the hypothesis that the activation of cyclooxygenase (COX) contributes to these alterations in bowel functions. DFU inhibition of COX‐2, but not SC‐560 inhibition of COX‐1, restored to normal levels the electrical properties of myenteric AH neurones, the proportion of S neurones exhibiting slow EPSPs, and the rate of propulsive motor activity. Neither inhibitor was effective in altering the level of inflammation, the increased availability of mucosal 5‐HT, or the enhanced fast EPSPs in myenteric AH and S neurones. COX‐2 expression is enhanced in the myenteric plexus and cells within the smooth muscle layers during colitis, possibly reflecting the site at which COX‐2 inhibition acts to allow recovery of motor function. In support of this concept, COX‐1, but not COX‐2, inhibition was effective in restoring normal mucosal prostaglandin levels. These results indicate that the various changes that occur in the motor neural pathways of the distal colon in TNBS‐induced colitis do not involve a single neuroimmune mechanism. COX‐2 activation is a critical step in the enhanced excitability of AH neurones as well as diminished propulsive motility in TNBS colitis, whereas other yet to be resolved pathways, that do not involve COX‐1 or COX‐2 activation, lead to altered 5‐HT content in the mucosa and an augmentation of fast EPSPs.

Antineuronal antibodies in idiopathic achalasia and gastro-oesophageal reflux disease

Background and aims: The precise aetiology of achalasia is unknown although autoimmunity has been implicated and is supported by several studies. We screened sera from patients with achalasia or gastro-oesophageal reflux disease (GORD) to test for circulating antimyenteric neuronal antibodies. Methods: Serum was obtained from 45 individuals with achalasia, 16 with GORD, and 22 normal controls. Serum was used in immunohistochemistry to label whole mount preparations of ileum and oesophagus of the guinea pig and mouse. Also, sections of superior cervical and dorsal root ganglia, and spinal cord were examined. Results: Positive immunostaining of the myenteric plexus was detected in significantly more achalasia and GORD samples than control samples (achalasia, p<0.001; GORD, p<0.01), and immunoreactivity was significantly more intense with achalasia and GORD serum samples than controls (achalasia, p<0.01; GORD, p<0.05). There was no correlation between intensity of immunoreactivity and duration of achalasia symptoms. In most cases, achalasia and GORD sera stained all ileal submucosal and myenteric neurones, and oesophageal neurones. Immunostaining was not species specific; however, immunostaining was largely specific for enteric neurones. Western blot analysis failed to reveal specific myenteric neuronal proteins that were labelled by antibodies in achalasia or GORD serum. Conclusions: These data suggest that antineuronal antibodies are generated in response to tissue damage or some other secondary phenomenon in achalasia and GORD. We conclude that antineuronal antibodies found in the serum of patients with achalasia represent an epiphenomenon and not a causative factor.

Distribution of adrenergic receptors in the enteric nervous system of the guinea pig, mouse, and rat

Adrenergic receptors in the enteric nervous system (ENS) are important in control of the gastrointestinal tract. Here we describe the distribution of adrenergic receptors in the ENS of the ileum and colon of the guinea pig, rat, and mouse by using single‐ and double‐labelling immunohistochemistry. In the myenteric plexus (MP) of the rat and mouse, α2a‐adrenergic receptors (α2a‐AR) were widely distributed on neurons and enteric glial cells. α2a‐AR mainly colocalized with calretinin in the MP, whereas submucosal α2a‐AR neurons colocalized with vasoactive intestinal polypeptide (VIP), neuropeptide Y, and calretinin in both species. In the guinea pig ileum, we observed widespread α2a‐AR immunoreactivity on nerve fibers in the MP and on VIP neurons in the submucosal plexus (SMP). We observed extensive β1‐adrenergic receptor (β1‐AR) expression on neurons and nerve fibers in both the MP and the SMP of all species. Similarly, the β2‐adrenergic receptor (β2‐AR) was expressed on neurons and nerve fibers in the SMP of all species, as well as in the MP of the mouse. In the MP, β1‐ and β2‐AR immunoreactivity was localized to several neuronal populations, including calretinin and nitrergic neurons. In the SMP of the guinea pig, β1‐ and β2‐AR mainly colocalized with VIP, whereas, in the rat and mouse, β1‐ and β2‐AR were distributed among the VIP and calretinin populations. Adrenergic receptors were widely localized on specific neuronal populations in all species studied. The role of glial α2a‐AR is unknown. These results suggest that sympathetic innervation of the ENS is directed toward both enteric neurons and enteric glia. J. Comp. Neurol. 495:529–553, 2006.

The subfornical organ: a central nervous system site for actions of circulating leptin

Adipose tissue plays a critical role in energy homeostasis, secreting adipokines that control feeding, thermogenesis, and neuroendocrine function. Leptin is the prototypic adipokine that acts centrally to signal long-term energy balance. While hypothalamic and brain stem nuclei are well-established sites of action of leptin, we tested the hypothesis that leptin signaling occurs in the subfornical organ (SFO). The SFO is a circumventricular organ (CVO) that lacks the normal blood-brain barrier, is an important site in central autonomic regulation, and has been suggested to have a role in modulating peripheral signals indicating energy status. We report here the presence of mRNA for the signaling form of the leptin receptor in SFO and leptin receptor localization by immunohistochemistry within this CVO. Central administration of leptin resulted in phosphorylation of STAT3 in neurons of SFO. Whole cell current-clamp recordings from dissociated SFO neurons demonstrated that leptin (10 nM) influenced the excitability of 64% (46/72) of SFO neurons. Leptin was found to depolarize the majority of responsive neurons with a mean change in membrane potential of 7.3 +/- 0.6 mV (39% of all SFO neurons), while the remaining cells that responded to leptin hyperpolarized (-6.9 +/- 0.7 mV, 25% of all SFO neurons). Similar depolarizing and hyperpolarizing effects of leptin were observed in recordings from acutely prepared SFO slice preparations. Leptin was found to influence the same population of SFO neurons influenced by amylin as three of four cells tested for the effects of bath application of both amylin and leptin depolarized to both peptides. These observations identify the SFO as a possible central nervous system location, with direct access to the peripheral circulation, at which leptin may act to influence hypothalamic control of energy homeostasis.

Inducible nitric oxide synthase (iNOS) in endotoxemia: chimeric mice reveal different cellular sources in various tissues

The aim of these experiments was to determine the contribution of leukocyte‐derived iNOS to total iNOS expression induced by lipopolysaccharide (LPS). By transferring bone marrow between iNOS+/+ and iNOS–/– mice, we created chimeric mice in which iNOS expression was limited to either circulating leukocytes (leukocyte‐iNOS mice) or parenchymal cells (parenchyma‐iNOS mice). Analysis of congenic markers demonstrated that >95% of thymocytes in chimeric mice were of donor origin. Also, following LPS treatment, iNOS mRNA was detectable in blood from leukocyte‐iNOS mice but not parenchyma‐iNOS mice. Together these findings indicated that the host marrow had been replaced entirely by donor cells. In the lung, at least 50% of the LPS‐induced iNOS mRNA was derived from leukocytes, and immunohistochemical analysis indicated that leukocytes were the main source of iNOS protein. In contrast in the liver, colon, and muscle, iNOS expression was derived predominantly from parenchymal cells. This divergence is potentially explained by the high level of leukocyte recruitment to the lung, relative to the other tissues. Plasma levels of NOS byproducts indicated that parenchymal iNOS was the dominant source of systemic iNOS activity. These findings indicate that in tissues other than the lung, parenchymal cells are the principal source of iNOS during endotoxemia.

Ionizing radiation reduces neurally evoked electrolyte transport in rat ileum through a mast cell-dependent mechanism.

BACKGROUND/AIMS Mechanisms of neuroimmune regulation of intestinal electrolyte transport under pathophysiological conditions are unclear. This study investigated the effect of ionizing radiation on ileal electrolyte transport. METHODS Rats were exposed to 10 Gy gamma-radiation and were killed 2, 24, and 48 hours later. Ileal segments were either mounted in Ussing chambers and exposed to electrical field stimulation, prostaglandin E2, leukotriene D4, or theophylline, or they were assayed for biochemical indices of inflammation. Other segments were processed for routine histological screening, mast cell counts, or immunohistochemical analysis of the distribution of vasoactive intestinal polypeptide or substance P immunoreactivity. RESULTS Basal short-circuit current was unchanged 2, 24, or 48 hours postirradiation. However, there was a reduction of tissue responsiveness to electrical field stimulation, prostaglandin E2, and theophylline but not to leukotriene D4. Decreased responsiveness at 2-hours postirradiation was blocked by pretreatment with the H1 antagonist pyrilamine. Tissue myeloperoxidase activity and 5-hydroxytryptamine content were not altered postirradiation, but tissue histamine and mucosal mast cells were significantly reduced at 24 and 48 hours. There were no significant changes in villus-crypt architecture until 48 hours postirradiation. There was no significant alteration in the distribution of immunoreactive vasoactive intestinal polypeptide or substance P. CONCLUSIONS Ionizing radiation reduced the transport response to neural stimulation. The effect correlated temporally with decreased mast cells and histamine, suggesting a functional role for previously reported mast cell-nerve interactions.

Inhibiting fatty acid amide hydrolase normalizes endotoxin-induced enhanced gastrointestinal motility in mice

BACKGROUND AND PURPOSE Gastrointestinal (GI) motility is regulated in part by fatty acid ethanolamides (FAEs), including the endocannabinoid (EC) anandamide (AEA). The actions of FAEs are terminated by fatty acid amide hydrolase (FAAH). We investigated the actions of the novel FAAH inhibitor AM3506 on normal and enhanced GI motility.

Substrate-Selective Inhibition of Cyclooxygenase-2: Development and Evaluation of Achiral Profen Probes

Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid and the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonoylethanolamide (AEA). We recently reported that (R)-profens selectively inhibit endocannabinoid oxygenation but not arachidonic acid oxygenation. In this work, we synthesized achiral derivatives of five profen scaffolds and evaluated them for substrate-selective inhibition using in vitro and cellular assays. The size of the substituents dictated the inhibitory strength of the analogs, with smaller substituents enabling greater potency but less selectivity. Inhibitors based on the flurbiprofen scaffold possessed the greatest potency and selectivity, with desmethylflurbiprofen (3a) exhibiting an IC50 of 0.11 μM for inhibition of 2-AG oxygenation. The crystal structure of desmethylflurbiprofen complexed to mCOX-2 demonstrated a similar binding mode to other profens. Desmethylflurbiprofen exhibited a half-life in mice comparable to that of ibuprofen. The data presented suggest that achiral profens can act as lead molecules toward in vivo probes of substrate-selective COX-2 inhibition.

Neuromuscular changes in a rat model of colitis

Intracolonic administration of Trichinella spiralis larvae in rats causes colitis with features similar to ulcerative colitis, notably with inflammation predominantly limited to the colonic mucosa. Our aim was to characterize the functional and neurochemical changes occurring within the myenteric (MP) and submucosal plexuses (SMP) during T. spiralis-induced colitis. Infected rats had decreased body weight, altered stool consistency and elevated myeloperoxidase activity, 6 and 14 days post-infection (PI). Responses to acetylcholine and KCl in circular muscle strips were reduced in infected tissues, demonstrating an impairment of contractility. In addition, there was a decrease in spontaneous motor activity and reduced sensitivity to the nitric oxide synthase (NOS) inhibitor L-NOArg, corresponding with a significant reduction in NOS immunoreactive neurons in the MP of infected animals. T. spiralis did not alter the total number of myenteric or submucosal neurons. Substance P innervation of submucosal blood vessels was reduced after infection, as were submucosal calretinin and calbindin immunoreactive neurons. No changes in choline acetyltransferase and calcitonin gene-related peptide immunoreactivity were observed. T. spiralis-induced colitis causes profound neuromuscular adaptations. The reduction in NOS neurons appears to underlie changes in motility.