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Serotonin [5-hydroxytryptamine (5-HT)] is released from enterochromaffin cells in the mucosa of the small intestine. We tested a hypothesis that elevation of 5-HT in the environment of enteric mast cells might degranulate the mast cells and release mediators that become paracrine signals to the enteric nervous system, spinal afferents, and secretory glands. Western blotting, immunofluorescence, ELISA, and pharmacological analysis were used to study expression of 5-HT receptors by mast cells in the small intestine and action of 5-HT to degranulate the mast cells and release histamine in guinea pig small intestine and segments of human jejunum discarded during Roux-en-Y gastric bypass surgeries. Mast cells in human and guinea pig preparations expressed the 5-HT1A receptor. ELISA detected spontaneous release of histamine in guinea pig and human preparations. The selective 5-HT1A receptor agonist 8-hydroxy-PIPAT evoked release of histamine. A selective 5-HT1A receptor antagonist, WAY-100135, suppressed stimulation of histamine release by 5-HT or 8-hydroxy-PIPAT. Mast cell-stabilizing drugs, doxantrazole and cromolyn sodium, suppressed the release of histamine evoked by 5-HT or 8-hydroxy-PIPAT in guinea pig and human preparations. Our results support the hypothesis that serotonergic degranulation of enteric mast cells and release of preformed mediators, including histamine, are mediated by the 5-HT1A serotonergic receptor. Association of 5-HT with the pathophysiology of functional gastrointestinal disorders (e.g., irritable bowel syndrome) underlies a question of whether selective 5-HT1A receptor antagonists might have therapeutic application in disorders of this nature.

Actions of lubiprostone, a selective type-2 chloride channel activator, on mucosal secretion were investigated in guinea pig small intestine and colon. Flat-sheet preparations were mounted in Ussing flux chambers for recording short-circuit current ( Isc) as a marker for electrogenic chloride secretion. Lubiprostone, applied to the small intestinal mucosa in eight concentrations ranging from 1–3000 nM, evoked increases in Isc in a concentration-dependent manner with an EC50 of 42.5 nM. Lubiprostone applied to the mucosa of the colon in eight concentrations ranging from 1–3000 nM evoked increases in Isc in a concentration-dependent manner with an EC50 of 31.7 nM. Blockade of enteric nerves by tetrodotoxin did not influence stimulation of Isc by lubiprostone. Antagonists acting at prostaglandin (PG)E2, EP1–3, or EP4 receptors did not suppress stimulation of Isc by lubiprostone but suppressed or abolished PGE2-evoked responses. Substitution of gluconate for chloride abolished all responses to lubiprostone. The selective CFTR channel blocker, CFTR(inh)-172, did not suppress lubiprostone-evoked Isc. The broadly acting blocker, glibenclamide, suppressed ( P < 0.001) lubiprostone-evoked Isc. Lubiprostone, in the presence of tetrodotoxin, enhanced carbachol-evoked Isc. The cholinergic component, but not the putative vasoactive intestinal peptide component, of neural responses to electrical field stimulation was enhanced by lubiprostone. Application of any of the prostaglandins, E2, F2, or I2, evoked depolarization of the resting membrane potential in enteric neurons. Unlike the prostaglandins, lubiprostone did not alter the electrical behavior of enteric neurons. Exposure to the histamine H2 receptor agonists increased basal Isc followed by persistent cyclical increases in Isc. Lubiprostone increased the peak amplitude of the dimaprit-evoked cycles.

Intracellular microelectrodes were used to record neurogenic inhibitory junction potentials in the intestinal circular muscle coat. Electrical field stimulation was used to stimulate intramural neurons and evoke contraction of the smooth musculature. Exposure to β-nicotinamide adenine dinucleotide (β-NAD) did not alter smooth muscle membrane potential in guinea pig colon or human jejunum. ATP, ADP, β-NAD, and adenosine, as well as the purinergic P2Y1 receptor antagonists MRS 2179 and MRS 2500 and the adenosine A1 receptor agonist 2-chloro- N6-cyclopentyladenosine, each suppressed inhibitory junction potentials in guinea pig and human preparations. β-NAD suppressed contractile force of twitch-like contractions evoked by electrical field stimulation in guinea pig and human preparations. P2Y1 receptor antagonists did not reverse this action. Stimulation of adenosine A1 receptors with 2-chloro- N6-cyclopentyladenosine suppressed the force of twitch contractions evoked by electrical field stimulation in like manner to the action of β-NAD. Blockade of adenosine A1 receptors with 8-cyclopentyl-1,3-dipropylxanthine suppressed the inhibitory action of β-NAD on the force of electrically evoked contractions. The results do not support an inhibitory neurotransmitter role for β-NAD at intestinal neuromuscular junctions. The data suggest that β-NAD is a ligand for the adenosine A1 receptor subtype expressed by neurons in the enteric nervous system. The influence of β-NAD on intestinal motility emerges from adenosine A1 receptor-mediated suppression of neurotransmitter release at inhibitory neuromuscular junctions.

Enteric neuroimmune interactions in gastrointestinal hypersensitivity responses involve antigen detection by mast cells, mast cell degranulation, release of chemical mediators, and modulatory actions of the mediators on the enteric nervous system (ENS). Electrophysiological methods were used to investigate electrical and synaptic behavior of neurons in the stomach and small intestine during exposure to β-lactoglobulin in guinea pigs sensitized to cow's milk. Application of β-lactoglobulin to sensitized preparations depolarized the membrane potential and increased neuronal excitability in small intestinal neurons but not in gastric neurons. Effects on membrane potential and excitability in the small intestine were suppressed by the mast cell stabilizing drug ketotifen, the histamine H2 receptor antagonist cimetidine, the cyclooxygenase inhibitor piroxicam, and the 5-lipoxygenase inhibitor caffeic acid. Unlike small intestinal ganglion cells, gastric myenteric neurons did not respond to histamine applied exogenously. Antigenic exposure suppressed noradrenergic inhibitory neurotransmission in the small intestinal submucosal plexus. The histamine H3receptor antagonist thioperamide and piroxicam, but not caffeic acid, prevented the allergic suppression of noradrenergic inhibitory neurotransmission. Antigenic stimulation of neuronal excitability and suppression of synaptic transmission occurred only in milk-sensitized animals. Results suggest that signaling between mast cells and the ENS underlies intestinal, but not gastric, anaphylactic responses associated with food allergies. Histamine, prostaglandins, and leukotrienes are paracrine signals in the communication pathway from mast cells to the small intestinal ENS.

Mast cells express the substance P (SP) neurokinin 1 receptor and the calcitonin gene-related peptide (CGRP) receptor in guinea pig and human small intestine. Enzyme-linked immunoassay showed that activation of intramural afferents by antidromic electrical stimulation or by capsaicin released SP and CGRP from human and guinea pig intestinal segments. Electrical stimulation of the afferents evoked slow excitatory postsynaptic potentials (EPSPs) in the enteric nervous system. The slow EPSPs were mediated by tachykinin neurokinin 1 and CGRP receptors. Capsaicin evoked slow EPSP-like responses that were suppressed by antagonists for protease-activated receptor 2. Afferent stimulation evoked slow EPSP-like excitation that was suppressed by mast cell-stabilizing drugs. Histamine and mast cell protease II were released by 1) exposure to SP or CGRP, 2) capsaicin, 3) compound 48/80, 4) elevation of mast cell Ca2+ by ionophore A23187, and 5) antidromic electrical stimulation of afferents. The mast cell stabilizers cromolyn and doxantrazole suppressed release of protease II and histamine when evoked by SP, CGRP, capsaicin, A23187, electrical stimulation of afferents, or compound 48/80. Neural blockade by tetrodotoxin prevented mast cell protease II release in response to antidromic electrical stimulation of mesenteric afferents. The results support a hypothesis that afferent innervation of enteric mast cells releases histamine and mast cell protease II, both of which are known to act in a diffuse paracrine manner to influence the behavior of enteric nervous system neurons and to elevate the sensitivity of spinal afferent terminals.

Background & Aims: Brain-derived neurotrophic factor (BDNF) may play a critical role in gut motility. We aimed to investigate BDNF's physiologic effects on gut motility in slowtransit constipation (STC) and to explore the underlying molecular mechanisms. Methods: BDNF expression and alterations of colonic nerve fiber density in STC patients were first investigated. BDNF's effects on gastrointestinal motility of both BDNF+/− mice and loperamide-induced constipation mice were then examined in vivo and in vitro. Smooth muscle α-actin (α-SMA) expression, and nerve fiber, neuromuscular junction (NMJ), and smooth muscle cell (SMC) alterations were investigated. Finally, the effects of BDNF-induced TrkBphospholipase C/inositol trisphosphate (TrkB-PLC/IP3) pathway activation on gut motility were investigated. Results: In STC patients, BDNF expression and nerve fiber density were decreased, and mucosal nerve fiber ultrastructural degenerations were demonstrated. Gut motility was decreased in vivo and in vitro in BDNF+/− and constipation mice, with BDNF dose-dependently increasing gut motility. In BDNF+/− mice, α-SMA expression and nerve fiber density were decreased, and nerve fiber, NMJ, and SMC ultrastructural degenerations were observed. Finally, TrkB-PLC/IP3 pathway agonists dramatically attenuated BDNF's excitatory effect on gut motility, and exogenous BDNF induced an obvious increase in IP3 expression. Conclusions: BDNF plays an important regulatory role in gut motility in STC. It was mediated by altering the intestinal innervation structure, as well as smooth muscle secondary degeneration through a mechanism involving TrkB-PLC/IP3 pathway activation.

The soluble guanylyl cyclase is expressed in guinea-pig gallbladder smooth muscle and agents that stimulate this enzyme activity cause gallbladder relaxation. The compound 5Cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine (BAY 41-2272) is a potent nitric oxide (NO)-independent soluble guanylyl cyclase stimulator, but little is known about its effects in gallbladder smooth muscle. This study investigated the mechanisms underlying the relaxations of guinea-pig gallbladder smooth muscle induced by BAY 41-2272. Gallbladder strips were mounted in 20-ml organ baths for isometric force recording. BAY 41-2272 concentration-dependently relaxed histamine-precontracted gallbladder strips. Prior incubation with the NO synthesis inhibitor L-NAME (0.1 mM) or the soluble guanylyl cyclase inhibitor ODQ (10 μM) caused significant rightward shifts in the concentration-response curves to BAY 41-2272. NO-donor, sodium nitroprusside caused concentration-dependent relaxations, which were greatly potentiated by BAY 41-2272 and almost completely inhibited by ODQ. To evaluate the effect of K+ channels, the relaxant responses of gallbladder muscle to Bay 41-2272 remained unchanged selective K+ channel inhibitor such as glibenclamide (10 μM), 4-aminopyridine (1 mM) or the co-incubation of charybdotoxin (0.1 μM) with apamin (1 μM). To evaluate the participation of prostanoids and cAMP pathway, the cyclooxygenase inhibitor indomethacin (10 μM) and selective phosphodiesterase type 4 inhibitor rolipram (1 μM) failed to significantly affect the BAY 41-2272-induced gallbladder relaxations. BAY 41-2272 shifted to the right the gallbladder contractile responses to either histamine (0.0001-10 μM). BAY 41-2272 (10 μM) also caused a marked rightward shift and decreased the maximal contractile responses to extracellular CaCl2. The effects of an inhibitor of the PKA, Rp-8CPT-cAMPS (10 μM), or the PKG, Rp8-pCPT-cGMPS (10 μM) on relaxation induced by the cumulative adminstration of BAY 41-2272 were studied in precontracted with 1 μM histamine. The relaxing effect of BAY 41-2272 were significantly attenuated by Rp-8-pCPT-cGMPS, but not by Rp-8CPT-cAMPS. These results indicate that BAY 41-2272 causes cGMP-dependent guinea-pig gallbladder smooth muscle relaxations in a synergistic fashion with NO. BAY 41-2272 has also an additional mechanism independently of soluble guanylyl cyclase activation possibly involving Ca2+ entry blockade.

G A A b st ra ct s effects on intestinal permeability. Methods: BALB/c (WT) and IL-13Rα1KO mice were infected with Heligmosomoides polygyrus and studied 12 days after a secondary infection (Hp2) to evoke a memory response. Muscle-free sections of jejunum were taken from WT or IL-13Rα1KO mice and mounted in microsnapwells to determine transepithelial electrical resistance (TEER), an index of mucosal permeability. Segments of jejunum were placed in Ussing chambers to assess changes in basal short circuit current (Isc). mRNA expression was determined by quantitative real-time PCR. Results: The Hp2-induced increase in Th2 cytokine expression was associated with decreased TEER (↑permeability) and basal Isc, consistent with enhanced paracellular permeability (Table). This was associated with an increase in mucosal mast cell protease-1 (mMCP-1) and claudin-2 expression (3.0 ± 0.7 fold). In contrast, Hp2 infection did not alter TEER or basal Isc in IL-13Rα1KO mice despite upregulation of Th2 cytokines, mMCP-1 and claudin-2 (3.5 ± 0.6 fold). Hp2 increased PAR2 expression in WT, but not in IL-13Rα1KO mice. Conclusions: These data question the perceived redundancy of the biological effects of IL-4 and IL-13. The effects of IL-4 on intestinal barrier function appear to be mediated by mastocytosis and increased expression of PAR-2 and claudin-2. In contrast, IL-13 working through IL-13Rα1 is critical for the Hp2induced changes in permeability, which appears to be due, in part, to increased expression of PAR-2, but independent of mastocytosis and claudin-2.

G A A b st ra ct s and involved in the various gut function. However little is known about the subpopulations of TRPV1-expressing nerves and sensory peptidergic nerves in gut. We previously demonstrated that TRPV1-positive nerve fibers in rectum were most abundant among the regions of mouse large intestine. In the present study, we investigated the immunohistochemical distribution of TRPV1 channels and sensory neuropeptides and their co-localization in mouse rectum. METHODS: Extensive TRPV1-immunoreactivity was detected by using immunohistochemical staining with fluorescein-conjugated tyramide amplification. Protein gene product 9.5 (PGP), CGRP, substance P (SP) and neurokinin A (NKA)-immunoreactivity was also detected by indirect staining with their specific antibodies. The longitudinal change in smooth muscle tone was isotonically measured using Magnus apparatus. RESULTS: The localization of TRPV1 was studied at both transverse and horizontal planes of sections in mouse rectum. Numerous TRPV1-immunoreactive nerve fibers were clearly detected in the mucosal, submucosal layer and myenteric plexus. In contrast, TRPV1 nerve fibers were sparsely distributed in circular and longitudinal muscle layers. Next, double labeling studies were carried out. In the mucosal layer, all the TRPV1 nerve fibers were found to colocalize with PGP and CGRP. SP and NKA positive nerve fibers were not observed. TRPV1 nerve fibers containing both CGRP and SP were observed running around blood vessels in the submucosal layer. In the myenteric plexus, most of the TRPV1 axons were found to colocalize with PGP and CGRP. Relatively small numbers of TRPV1 axons were immunopositive for SP. NKA and SP-immunoreactive axons were observed in the circular and longitudinal muscle, and the cell bodies were detected in the myenteric plexus. NKA and SP were almost colocalized at the axons and cell bodies in muscle layer. In the Magnus experiment, capsaicininduced contraction was significantly inhibited by NK1 and NK2 receptor antagonists. DISCUSION: The present results suggest that TRPV1 nerve fibers in mouse rectum are extrinsic primary afferents. TRPV1 fibers containing CGRP are distributed in mucosal, submucosal layer and myenteric plexus and play various roles in rectal function. TRPV1 fibers containing SP and/or NKA are distributed in myenteric plexus and involved in rectal motor function by stimulating intrinsic excitatory motor neurons.

G A A b st ra ct s and involved in the various gut function. However little is known about the subpopulations of TRPV1-expressing nerves and sensory peptidergic nerves in gut. We previously demonstrated that TRPV1-positive nerve fibers in rectum were most abundant among the regions of mouse large intestine. In the present study, we investigated the immunohistochemical distribution of TRPV1 channels and sensory neuropeptides and their co-localization in mouse rectum. METHODS: Extensive TRPV1-immunoreactivity was detected by using immunohistochemical staining with fluorescein-conjugated tyramide amplification. Protein gene product 9.5 (PGP), CGRP, substance P (SP) and neurokinin A (NKA)-immunoreactivity was also detected by indirect staining with their specific antibodies. The longitudinal change in smooth muscle tone was isotonically measured using Magnus apparatus. RESULTS: The localization of TRPV1 was studied at both transverse and horizontal planes of sections in mouse rectum. Numerous TRPV1-immunoreactive nerve fibers were clearly detected in the mucosal, submucosal layer and myenteric plexus. In contrast, TRPV1 nerve fibers were sparsely distributed in circular and longitudinal muscle layers. Next, double labeling studies were carried out. In the mucosal layer, all the TRPV1 nerve fibers were found to colocalize with PGP and CGRP. SP and NKA positive nerve fibers were not observed. TRPV1 nerve fibers containing both CGRP and SP were observed running around blood vessels in the submucosal layer. In the myenteric plexus, most of the TRPV1 axons were found to colocalize with PGP and CGRP. Relatively small numbers of TRPV1 axons were immunopositive for SP. NKA and SP-immunoreactive axons were observed in the circular and longitudinal muscle, and the cell bodies were detected in the myenteric plexus. NKA and SP were almost colocalized at the axons and cell bodies in muscle layer. In the Magnus experiment, capsaicininduced contraction was significantly inhibited by NK1 and NK2 receptor antagonists. DISCUSION: The present results suggest that TRPV1 nerve fibers in mouse rectum are extrinsic primary afferents. TRPV1 fibers containing CGRP are distributed in mucosal, submucosal layer and myenteric plexus and play various roles in rectal function. TRPV1 fibers containing SP and/or NKA are distributed in myenteric plexus and involved in rectal motor function by stimulating intrinsic excitatory motor neurons.

Introduction: Patients with IBD show higher thresholds to slow ramp distension of the rectum, as opposed to patients with a diarrhea-predominant IBS (IBS-D). Recently we have found elevated serine protease (SerP) activities in both IBS-D and ulcerative colitis (UC) fecal materials. In mice, intracolonic (IC) infusion of fecal IBS-D supernatants evokes a proteinase-activated receptor (PAR)-2 mediated colonic hypersensitivity to distension. Objectives: Our aims were to evaluate the influence of fecal supernatant from UC patients on visceral sensitivity in mice and to test the involvement of PAR-4, and its activator cathepsin G (cat-G) as possible mediators in this nociceptive response. Methods: Fecal samples from UC patients and healthy subjects were dissolved in saline, centrifuged and filtered. Under anaesthesia electrodes were inserted in the abdominal muscle to record abdominal cramps in C57/BL6 male mice. Four days after surgery mice were IC infused with 0.3 mL fecal supernatant from UC patients or controls, and colorectal distensions (CRD) were performed using a balloon inflated from 0 to 0.12 mL each steps lasting 10 sec with 5 min intervals. To evaluate the role of PAR-4, micewere treatedwith a PAR-4 antagonist (P4pal-10, pepducin, 3x0.25 mg/kg) IP before and during UC supernatant infusion. The participation of cat-G was assessed by incubating the UC supernatant with a mixture of antiproteases (aprotinin and SBTI, 0.22 and 1.70 mg/mL respectively) or with a cat-G inhibitor (0.057 mg/mL). The effect of PAR-4 activation or cat-G was tested by IC infusion of 100 ug PAR-4 agonist (PAR4AP, 0.667mg/mL) or 0.025 UN cat-G (0.167UN/mL). Results: IC infusion of UC supernatants decreased the intensity of abdominal EMG response by 71%, 49% and 35% for 0.04, 0.06 and 0.08 mL of CRD (p

10). CCK sensitive currents are also abolished by 2-APB, ruthenium red and SKF96365. Widespread immunoreactivity was seen for all TRPC subunits. Conclusions: NGNs exhibit currents activated by known agonists of TRPC channels. The OAG activated current most closely resembles TRPC1 with near linear I/V relationship. However the current sensitive to the nonselective TRPC blockers 2-APB and SKF96365 is more complex, likely reflecting a composite of TRPC1 and possibly TRPC4 and / or 5, as these subunits can heterodimerize. The presence of currents similar to TRPC1 is relevant for gastrointestinal vagal afferents, as this channel is known to also be mechanosensitive. TRPC channels may integrate the complex chemical and possibly mechanical stimuli which activate vagal afferents.

We tested a hypothesis that silencing of CRF expression in the enteric nervous system (ENS) attenuates stress-evoked alterations of colonic motor function. Laser capture microdissection (LCM), real time RT-PCR, and immunofluorescence were used to study expression of CRF in the ENS of the rat, mouse and guinea pig colon. Injection of double-stranded RNA (dsRNA) for CRF (dsCRF, 20 μg/rat) into the rat colon was used to achieve RNA interference (RNAi)-mediated inhibition of CRF gene expression. DsRNA for β-globulin was a control (dsControl). Four days after dsRNA administration, rats were exposed to restraint stress for 2 h. Fecal output was monitored at 15 min intervals for 2 h. LCM and RT-PCR revealed CRF mRNA expression in myenteric neurons in rat proximal and distal colon. CRF-like immunoreactivity (IR) was expressed in nerve cell bodies and fibers in the myenteric and submucosal plexuses of the rat, mouse, and guinea pig colon. CRF-IR neurons were 5.4±0.3% of the myenteric population and 21.9±4.5% of the submucosal population in rat colon. In rat colonic myenteric plexus, 45.0±10.5% of CRF-IR neurons expressed choline acetyltransferase-IR and 97.1±1.4% expressed nitric oxide synthase-IR. In the rat colonic submucosal plexus, all CRF-IR neurons expressed vasoactive intestinal peptide-IR. Stress induced widespread immunostaining for c-fos in colonic myenteric ganglia. Double labeling showed that most of the c-fos-positive neurons in stressed animals expressed either CRF-IR or CRF1receptor-IR. Intramural injection of dsRNA for CRF (dsCRF) “knocked-down” basal CRF expression in rat colon, while CRF expression in dsControl animals was unaffected. CRF mRNA was decreased by 60% after four days. CRF peptide-IR was undetectable or very low in myenteric and submucosal neurons of the rat colon. DsControl and dsCRFtreated rats had similar daily food intake, body weight gain, and cumulative fecal output during the 4 days post injection. In dsControl-treated rats, restraint stress induced a significant increase in fecal output when compared to non-stressed controls (stress: 9.7 ± 1.3 vs. nonstress: 2.7 ± 0.8; P 0.05). Stress changes CRF expression in the colonic ENS and is a factor in stress-evoked colon dysmotility. (Support: NIH R01 DK37238 and R01 DK57075 (JD Wood), PhRMA Research Starter Award (S Liu), UNC Center for Functional GI & Motility Disorders seed grant (S Liu), and Hellman Grant (A Bhargava).