Guinea Pig Distal Colon Research Articles

The presence of 5‐HT in myenteric varicosities is not due to uptake of 5‐HT released from the mucosa during dissection: use of a novel method for quantifying 5‐HT immunoreactivity in myenteric ganglia

Quantifying the relative abundance of different neurotransmitters in the myenteric plexus has proved challenging using conventional immunocytochemical approaches. Here, we present a new method of quantifying neurotransmitter content of an important enteric signalling molecule, serotonin (5-HT), in the myenteric plexus of guinea pig colon under different experimental conditions. Sections of guinea pig distal colon were exposed to different conditions including changes in temperature, dissection protocol, stimulation with faecal pellet distension and exogenous 5-HT. Sections were fixed and immuno-labelled for 5-HT. 5-HT staining density was quantified within myenteric plexus ganglia using defined settings and an analysis approach that uses threshold settings allowing for variances in background and tissue staining intensities and which calculates the area of tissue containing 5-HT above these thresholds. No differences were found in 5-HT immunoreactivity in the myenteric plexus when compared between tissues that were freshly fixed, undissected, or with mucosa and submucous plexus dissected away at either 4 or 37°C. Increased myenteric plexus 5-HT density was observed in preparations repeatedly stimulated using faecal pellet stimulation prior to fixation. Furthermore, exogenous 5-HT also increased 5-HT density. We demonstrate that quantitative differences in 5-HT immunoreactivity can be characterized using immunohistochemistry. This approach may be applied to measuring other neurotransmitter(s) within the enteric nervous system. While 5-HT is present in the guinea-pig enteric ganglia, this is not due to accumulation via in vitro handling and release from the mucosa, and furthermore, repeated colonic stimulation via distension increases 5-HT in the myenteric plexus.

Nitric oxide regulates polarity of guinea pig distal colon pellet propagation and circular muscle motor response

Peristaltic reflex does not fully explain the polarity of peristalsis. In the distal colon, we propose that the balance of circular muscle contraction proximal and distal to the pellet is a local reflex that predicts polarity of peristalsis. Guinea pig distal colon segments were harvested, and fecal pellet transit was measured. Photographs of fecal pellet propagation were taken, colonic radius proximal and distal to the moving pellet was measured, and the ratio of radius proximal to pellet to radius distal to pellet was measured. Isometric transducers were attached 1 cm proximal and distal to a fixed intraluminal balloon, and circular muscle (CM) response to balloon distension was recorded. N (G)-Nitro-L-arginine methyl ester hydrochloride (L-NAME) was used to elicit an effect in experimental preparations and compared to controls. Fecal pellet transit was delayed after L-NAME treatment (163 ± 23.9 vs. 41.5 ± 1.9 s in control, n = 6, p < 0.001). Photo-analysis revealed a ratio of proximal/distal colonic radius in control colon to be 0.72 ± 0.02 (n = 17) and in L-NAME treated colon 1.0 ± 0.02 (n = 10, p < 0.01). The dominant CM response pattern was a large contraction proximal to the inflated balloon and a smaller contraction distal to the balloon. In the presence of L-NAME, distal contraction was larger than the proximal contraction in 66% of the experiments. Pellet propulsion in the guinea pig's distal colon depends on nitric oxide to provide appropriate balance of force between proximal and distal contraction, resulting in pellet propagation toward the anus. CM contracts both proximal and distal to the pellet, and the polarity of pellet progression depends on the balance of the two forces.

Activation of electrogenic K secretion requires BK channels in guinea pig distal colon

A variety of hormones and neurotransmitters activate electrogenic K+ secretion in guinea pig distal colon, generally together with Cl− secretion. Blockers of BK channels (KCa1.1, Kcnma1), iberiotoxin (IbTx) and paxilline, inhibited the short‐circuit current (Isc) associated with K+ secretion. Consistent with this K+ secretion occurring via apical membrane BK, mucosal addition of IbTx inhibited epinephrine (epi) activation with an IC50 for Isc (epiIsc) and for transepithelial conductance (epiGt) of ~200 nM. However, maximal inhibition was only ~50%. Mucosally added paxilline [10 μM] also inhibited epiIsc and epiGt by ~50%. IbTx and paxilline each inhibited Isc activated by mucosal ATP, supporting apical BK as an absolute requirement for this K+ secretion. Sensitivity to IbTx and paxilline demonstrated K+ secretion during activation of Cl− secretion by prostaglandin‐E2 and a cholinergic agonist. Distal colonic epithelial cells expressed BKα mRNA with the ZERO splice variant and 3 splice variants for the C‐terminus. These cells also expressed the regulatory β‐subunits BKβ1 and BKβ4. Immuno‐localization demonstrated BKα in apical and basolateral membranes of surface and crypt cells. Together these results support a cellular mechanism for electrogenic K+ secretion involving activation of apical membrane BK, but epi activated K+ secretion also required opening of other K+ channel types. [NIH DK65845]

Is serotonin in enteric nerves required for distension-evoked peristalsis and propulsion of content in guinea-pig distal colon?

Recent studies have shown genetic deletion of the gene that synthesizes 5-HT in enteric neurons (tryptophan hydroxylase-2, Tph-2) leads to a reduction in intestinal transit. However, deletion of the Tph-2 gene also leads to major developmental changes in enteric ganglia, which could also explain changes in intestinal transit. We sought to investigate this further by acutely depleting serotonin from enteric neurons over a 24-h period, without the confounding influences induced by genetic manipulation. Guinea-pigs were injected with reserpine 24h prior to euthanasia. Video-imaging and spatio-temporal mapping was used to record peristalsis evoked by natural fecal pellets, or slow infusion of intraluminal fluid. Immunohistochemical staining for 5-HT was used to detect the presence of serotonin in the myenteric plexus. It was found that endogenous 5-HT was always detected in myenteric ganglia of control animals, but never in guinea-pigs treated with reserpine. Interestingly, peristalsis was still reliably evoked by either intraluminal fluid, or fecal pellets in reserpine-treated animals that also had their entire mucosa and submucosal plexus removed. In these 5-HT depleted animals, there was no change in the frequency of peristalsis or force generated during peristalsis. In control animals, or reserpine treated animals, high concentrations (up to 10μM) of ondansetron and SDZ-205-557, or granisetron and SDZ-205-557 had no effect on peristalsis. In summary, acute depletion of serotonin from enteric nerves does not prevent distension-evoked peristalsis, nor propulsion of luminal content. Also, we found no evidence that 5-HT3 and 5-HT4 receptor activation is required for peristalsis, or propulsion of contents to occur. Taken together, we suggest that the intrinsic mechanisms that generate peristalsis and entrain propagation along the isolated guinea-pig distal colon are independent of 5-HT in enteric neurons or the mucosa, and do not require the activation of 5-HT3 or 5-HT4 receptors.

5-HT3 and 5-HT4 antagonists inhibit peristaltic contractions in guinea-pig distal colon by mechanisms independent of endogenous 5-HT

Recent studies have shown that endogenous serotonin is not required for colonic peristalsis in vitro, nor gastrointestinal (GI) transit in vivo. However, antagonists of 5-Hydroxytryptamine (5-HT) receptors can inhibit peristalsis and GI-transit in mammals, including humans. This raises the question of how these antagonists inhibit GI-motility and transit, if depletion of endogenous 5-HT does not cause any significant inhibitory changes to either GI-motility or transit? We investigated the mechanism by which 5-HT3 and 5-HT4 antagonists inhibit distension-evoked peristaltic contractions in guinea-pig distal colon. In control animals, repetitive peristaltic contractions of the circular muscle were evoked in response to fixed fecal pellet distension. Distension-evoked peristaltic contractions were unaffected in animals with mucosa and submucosal plexus removed, that were also treated with reserpine (to deplete neuronal 5-HT). In control animals, peristaltic contractions were blocked temporarily by ondansetron (1–10 μM) and SDZ-205–557 (1–10 μM) in many animals. Interestingly, after this temporary blockade, and whilst in the continued presence of these antagonists, peristaltic contractions recovered, with characteristics no different from controls. Surprisingly, similar effects were seen in mucosa-free preparations, which had no detectable 5-HT, as detected by mass spectrometry. In summary, distension-evoked peristaltic reflex contractions of the circular muscle layer of the guinea-pig colon can be inhibited temporarily, or permanently, in the same preparation by selective 5-HT3 and 5-HT4 antagonists, depending on the concentration of the antagonists applied. These effects also occur in preparations that lack any detectable 5-HT. We suggest caution should be exercised when interpreting the effects of 5-HT3 and 5-HT4 antagonists; and the role of endogenous 5-HT, in the generation of distension-evoked colonic peristalsis.

Role of the BK channel (KCa1.1) during activation of electrogenic K+ secretion in guinea pig distal colon

Secretagogues acting at a variety of receptor types activate electrogenic K(+) secretion in guinea pig distal colon, often accompanied by Cl(-) secretion. Distinct blockers of K(Ca)1.1 (BK, Kcnma1), iberiotoxin (IbTx), and paxilline inhibited the negative short-circuit current (I(sc)) associated with K(+) secretion. Mucosal addition of IbTx inhibited epinephrine-activated I(sc) ((epi)I(sc)) and transepithelial conductance ((epi)G(t)) consistent with K(+) secretion occurring via apical membrane K(Ca)1.1. The concentration dependence of IbTx inhibition of (epi)I(sc) yielded an IC(50) of 193 nM, with a maximal inhibition of 51%. Similarly, IbTx inhibited (epi)G(t) with an IC(50) of 220 nM and maximal inhibition of 48%. Mucosally added paxilline (10 μM) inhibited (epi)I(sc) and (epi)G(t) by ∼50%. IbTx and paxilline also inhibited I(sc) activated by mucosal ATP, supporting apical K(Ca)1.1 as a requirement for this K(+) secretagogue. Responses to IbTx and paxilline indicated that a component of K(+) secretion occurred during activation of Cl(-) secretion by prostaglandin-E(2) and cholinergic stimulation. Analysis of K(Ca)1.1α mRNA expression in distal colonic epithelial cells indicated the presence of the ZERO splice variant and three splice variants for the COOH terminus. The presence of the regulatory β-subunits K(Ca)β1 and K(Ca)β4 also was demonstrated. Immunolocalization supported the presence of K(Ca)1.1α in apical and basolateral membranes of surface and crypt cells. Together these results support a cellular mechanism for electrogenic K(+) secretion involving apical membrane K(Ca)1.1 during activation by several secretagogue types, but the observed K(+) secretion likely required the activity of additional K(+) channel types in the apical membrane.

Identification and mechanosensitivity of viscerofugal neurons

Enteric viscerofugal neurons are interneurons with cell bodies in the gut wall; they project to prevertebral ganglia where they provide excitatory synaptic drive to sympathetic neurons which control intestinal motility and secretion. Here, we studied the mechanosensitivity and firing of single, identified viscerofugal neurons in guinea-pig distal colon. Flat sheet preparations of gut were set up in vitro and conventional extracellular recordings made from colonic nerve trunks. The nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) (1mM), was locally pressure ejected onto individual myenteric ganglia. In a few ganglia, DMPP promptly evoked firing in colonic nerves. Biotinamide filling of colonic nerves revealed that DMPP-responsive sites corresponded to viscerofugal nerve cell bodies. This provides a robust means to positively identify viscerofugal neuron firing. Of 15 single units identified in this way, none responded to locally-applied capsaicin (1μM). Probing with von Frey hairs at DMPP-responsive sites reliably evoked firing in all identified viscerofugal neurons (18/18 units tested; 0.8–5mN). Circumferential stretch of the preparation increased firing in all 14/14 units (1–5g, p<0.05). Both stretch and von Frey hair responses persisted in Ca2+-free solution (6mMMg2+, 1mM EDTA), indicating that viscerofugal neurons are directly mechanosensitive. To investigate their adequate stimulus, circular muscle tension and length were independently modulated (BAY K8644, 1μM and 10μM, respectively). Increases in intramural tension without changes in length did not affect firing. However, contraction-evoked shortening, under constant load, significantly decreased firing (p<0.001). In conclusion, viscerofugal neuron action potentials contribute to recordings from colonic nerve trunks, in vitro. They provide a significant primary afferent output from the colon, encoding circumferential length, largely independent of muscle tension. All viscerofugal neurons are directly mechanosensitive, although they have been reported to receive synaptic inputs. In short, viscerofugal neurons combine interneuronal function with length-sensitive mechanosensitivity.

Viscerofugal neurons recorded from guinea‐pig colonic nerves after organ culture

Enteric viscerofugal neurons provide cholinergic synaptic inputs to prevertebral sympathetic neurons, forming reflex circuits that control motility and secretion. Extracellular recordings of identified viscerofugal neurons have not been reported. Preparations of guinea pig distal colon were maintained in organotypic culture for 4-6 days (n = 12), before biotinamide tracing, immunohistochemistry, or extracellular electrophysiological recordings from colonic nerves. After 4-6 days in organ culture, calcitonin gene-related peptide and tyrosine hydroxylase immunoreactivity in enteric ganglia was depleted, and capsaicin-induced firing (0.4 μmol L(-1) ) was not detected, indicating that extrinsic sympathetic and sensory axons degenerate in organ culture. Neuroanatomical tracing of colonic nerves revealed that viscerofugal neurons persist and increase as a proportion of surviving axons. Extracellular recordings of colonic nerves revealed ongoing action potentials. Interestingly, synchronous bursts of action potentials were seen in 10 of 12 preparations; bursts were abolished by hexamethonium, which also reduced firing rate (400 μmol L(-1) , P < 0.01, n = 7). DMPP (1,1-dimethyl-4-phenylpiperazinium; 10(-4) mol L(-1) ) evoked prolonged action potential discharge. Increased firing preceded both spontaneous and stretch-evoked contractions (χ(2) = 11.8, df = 1, P < 0.001). Firing was also modestly increased during distensions that did not evoke reflex contractions. All single units (11/11) responded to von Frey hairs (100-300 mg) in hexamethonium or Ca(2+) -free solution. Action potentials recorded from colonic nerves in organ cultured preparations originated from viscerofugal neurons. They receive nicotinic input, which coordinates ongoing burst firing. Large bursts preceded spontaneous and reflex-evoked contractions, suggesting their synaptic inputs may arise from enteric circuitry that also drives motility. Viscerofugal neurons were directly mechanosensitive to focal compression by von Frey hairs.

Membrane trafficking produces distinct beta‐adrenergic signaling for Cl secretion and K secretion in guinea pig distal colonic mucosa

Epinephrine (epi) activated Cl and K secretion (sec) in isolated mucosa from guinea pig distal colon, measured as short‐circuit current. These components of the response were distinguished by inhibition of Cl sec with a β2 antagonist (ICI‐118551) that spared K sec; propranolol inhibited both. Interfering with endocytosis prolonged the transient Cl sec and inhibited sustained K sec. Both the dynamin inhibitor dynasore and the clathrin‐coated‐pit blocker mono‐dansylcadaverine produced this response. Dynasore also inhibited the sustained epi‐induced increase in mucosal cAMP, while leaving the transient cAMP increase intact. Manipulating the ubiquitylation cycle by inhibiting ubiquitin‐E1 ligase (UBEI‐41, BioGenova) also prolonged the epi‐induced Cl sec transient and slowed the onset of K sec. Conversely, inhibiting deubiquitylases (PR‐619, LifeSensors) alone stimulated K sec, while blunting Cl sec and producing a non‐additive increase in K sec with epi. Disruption of the actin (latrunculin B, cytochalasin D) and microtubule (nocodazole) cytoskeleton inhibited epi‐induced K sec without altering Cl sec. Together these results support an activation process involving internalization of β2‐adrenergic receptors that terminates signaling for Cl sec, and β‐adrenergic stimulation of K sec that requires receptor internalization prior to activation of the sustained cAMP signaling cascades. [NIH DK65845]

Activation of colonic mucosal 5-HT(4) receptors accelerates propulsive motility and inhibits visceral hypersensitivity.

5-hydroxytryptamine receptor (5-HT(4)R) agonists promote gastrointestinal motility and attenuate visceral pain, but concerns about adverse reactions have restricted their availability. We tested the hypotheses that 5-HT(4) receptors are expressed in the colonic epithelium and that 5-HT(4)R agonists can act intraluminally to increase motility and reduce visceral hypersensitivity. Mucosal expression of the 5-HT(4)R was evaluated by reverse-transcriptase polymerase chain reaction and immunohistochemical analysis of tissues from 5-HT(4)R(BAC)-enhanced green fluorescent protein mice. Amperometry, histology, and short-circuit current measurements were used to study 5-HT, mucus, and Cl(-) secretion, respectively. Propulsive motility was measured in guinea pig distal colon, and visceromotor responses were recorded in a rat model of colonic hypersensitivity. 5-HT(4)R compounds included cisapride, tegaserod, naronapride, SB204070, and GR113808. Mucosal 5-HT(4) receptors were present in the small and large intestines. In the distal colon, 5-HT(4) receptors were expressed by most epithelial cells, including enterochromaffin and goblet cells. Stimulation of 5-HT(4)Rs evoked mucosal 5-HT release, goblet cell degranulation, and Cl(-) secretion. Luminal administration of 5-HT(4)R agonists accelerated propulsive motility; a 5-HT(4)R antagonist blocked this effect. Bath application of 5-HT(4)R agonists did not affect motility. Oral or intracolonic administration of 5-HT(4)R agonists attenuated visceral hypersensitivity. Intracolonic administration was more potent than oral administration, and was inhibited by a 5-HT(4)R antagonist. Mucosal 5-HT(4) receptor activation can mediate the prokinetic and antinociceptive actions of 5-HT(4)R agonists. Colon-targeted, intraluminal delivery of 5-HT(4)R agonists might be used to promote motility and alleviate visceral pain, while restricting systemic bioavailability and resulting adverse side effects.

Mechanisms underlying distension-evoked peristalsis in guinea pig distal colon: is there a role for enterochromaffin cells?

The mechanisms underlying distension-evoked peristalsis in the colon are incompletely understood. It is well known that, following colonic distension, 5-hydroxytryptamine (5-HT) is released from enterochromaffin (EC) cells in the intestinal mucosa. It is also known that exogenous 5-HT can stimulate peristalsis. These observations have led some investigators to propose that endogenous 5-HT release from EC cells might be involved in the initiation of colonic peristalsis, following distension. However, because no direct evidence exists to support this hypothesis, the aim of this study was to determine directly whether release of 5-HT from EC cells was required for distension-evoked colonic peristalsis. Real-time amperometric recordings of 5-HT release and video imaging of colonic wall movements were performed on isolated segments of guinea pig distal colon, during distension-evoked peristalsis. Amperometric recordings revealed basal and transient release of 5-HT from EC cells before and during the initiation of peristalsis, respectively. However, removal of mucosa (and submucosal plexus) abolished 5-HT release but did not inhibit the initiation of peristalsis nor prevent the propagation of fecal pellets or intraluminal fluid. Maintained colonic distension by fecal pellets induced repetitive peristaltic waves, whose intrinsic frequency was also unaffected by removal of the submucosal plexus and mucosa, although their propagation velocities were slower. In conclusion, the mechanoreceptors and sensory neurons activated by radial distension to initiate peristalsis lie in the myenteric plexus and/or muscularis externa, and their activation does not require the submucosal plexus, release of 5-HT from EC cells, nor the presence of the mucosa. The propagation of peristalsis and propulsion of liquid or solid content along the colon is entrained by activity within the myenteric plexus and/or muscularis externa and does not require sensory feedback from the mucosa, nor neural inputs arising from submucosal ganglia.

Basolateral membrane Cl conductance in guinea pig distal colon activates during electrogenic K secretion and Cl secretion

Epinephrine (epi) activates transient Cl secretion (sec) and sustained K sec across isolated distal colonic mucosa of guinea pig. The Ca-activated Cl channel inhibitor CaCCinh-A01 [30μM] reduced electrogenic K sec, detected as short-circuit current (Isc), consistent with basolateral Cl channels providing an exit pathway for Cl entering via Na/K/2Cl-cotransporters. CaCCinh-A01 inhibited both Isc and transepithelial conductance in a concentration dependent manner, IC50 = 6.3μM. GlyH-101, another Cl channel inhibitor, also reduced K secretory Isc (IC50 = 9.4μM). Epi activated whole-cell Cl current in isolated intact colonic crypts. This epi-activated Cl current also was inhibited by CaCCinh-A01 or GlyH-101. In contrast to K sec, CaCCinh-A01 augmented epi-activated Cl secretory Isc as well as PGE2-activated Cl sec. Synergistic Cl sec activated by cholinergic/PGE2 stimulation was insensitive to CaCCinh-A01. Colonic expression of Tmem16A, a Ca-activated Cl channel, was supported by RT-PCR of Tmem16A-mRNA, immuno-blot with Tmem16A-antibodies, and immuno-reactivity in lateral membranes of epithelial cells. Alternative splices of Tmem16A were detected for exons involved in channel activation. Inhibition of K sec and augmentation of Cl sec by CaCCinh-A01 supports a common colonic cell model for these two ion secretory processes with basolateral membrane Cl channels that contribute to production of electrogenic K sec and limiting Cl sec. Maximal Cl sec occurs only for synergistic activation mechanisms that inactivate these basolateral membrane Cl channels. [NIH DK65845]

The relationship between inflammation-induced neuronal excitability and disrupted motor activity in the guinea pig distal colon

Colitis is associated with increased excitability of afterhyperpolarization neurons (AH neurons) and facilitated synaptic transmission in the myenteric plexus. These changes are accompanied by disrupted propulsive motility, particularly in ulcerated regions. This study examined the relationship between myenteric AH neuronal hyperexcitability and disrupted propulsive motility. The voltage-activated Na(+) channel opener veratridine, the intermediate conductance Ca(2+) -activated K(+) channel inhibitor TRAM-34 and the 5-HT(4) receptor agonist tegaserod were used to evaluate the effects of neuronal hyperexcitability and synaptic facilitation on propulsive motility in normal guinea pig distal colon. Because trinitrobenzene sulfonic acid (TNBS)-colitis-induced hyperexcitability of myenteric afferent neurons involves increases in hyperpolarization-activated, cyclic nucleotide-gated (HCN) channel activity, the HCN channel inhibitors Cs(+) and ZD7288 were used to suppress AH neuronal activity in TNBS-colitis. In non-inflamed preparations, veratridine halted propulsive motility (P<0.001). The rate of propulsive motor activity was significantly reduced following addition of TRAM-34 and tegaserod (P<0.001). In TNBS-inflamed preparations, in which motility was temporarily halted or obstructed at sites of ulceration, both Cs(+) and ZD7288 normalized motility through the inflamed regions. Immunohistochemistry studies demonstrated that the proportion of AH neurons in the myenteric plexus was unchanged in ulcerated regions, but there was a 10% reduction in total number of neurons per ganglion. These findings support the concept that inflammation-induced neuroplasticity in myenteric neurons, involving changes in ion channel activity that lead to enhanced AH neuronal excitability, can contribute to impaired propulsive colonic motility.

Gastrointestinal Motility Monitor (GIMM)

The Gastrointestinal Motility Monitor (GIMM; Catamount Research and Development; St. Albans, VT) is an in vitro system that monitors propulsive motility in isolated segments of guinea pig distal colon. The complete system consists of a computer, video camera, illuminated organ bath, peristaltic and heated water bath circulating pumps, and custom GIMM software to record and analyze data. Compared with traditional methods of monitoring colonic peristalsis, the GIMM system allows for continuous, quantitative evaluation of motility. The guinea pig distal colon is bathed in warmed, oxygenated Krebs solution, and fecal pellets inserted in the oral end are propelled along the segment of colon at a rate of about 2 mm/sec. Movies of the fecal pellet proceeding along the segment are captured, and the GIMM software can be used track the progress of the fecal pellet. Rates of propulsive motility can be obtained for the entire segment or for any particular region of interest. In addition to analysis of bolus-induced motility patterns, spatiotemporal maps can be constructed from captured video segments to assess spontaneous motor activity patterns. Applications of this system include pharmacological evaluation of the effects of receptor agonists and antagonists on propulsive motility, as well as assessment of changes that result from pathophysiological conditions, such as inflammation or stress. The guinea pig distal colon propulsive motility assay, using the GIMM system, is straightforward and simple to learn, and it provides a reliable and reproducible method of assessing propulsive motility.

Gastrointestinal Motility Monitor (GIMM)

The Gastrointestinal Motility Monitor (GIMM; Catamount Research and Development; St. Albans, VT) is an in vitro system that monitors propulsive motility in isolated segments of guinea pig distal colon. The complete system consists of a computer, video camera, illuminated organ bath, peristaltic and heated water bath circulating pumps, and custom GIMM software to record and analyze data. Compared with traditional methods of monitoring colonic peristalsis, the GIMM system allows for continuous, quantitative evaluation of motility. The guinea pig distal colon is bathed in warmed, oxygenated Krebs solution, and fecal pellets inserted in the oral end are propelled along the segment of colon at a rate of about 2 mm/sec. Movies of the fecal pellet proceeding along the segment are captured, and the GIMM software can be used track the progress of the fecal pellet. Rates of propulsive motility can be obtained for the entire segment or for any particular region of interest. In addition to analysis of bolus-induced motility patterns, spatiotemporal maps can be constructed from captured video segments to assess spontaneous motor activity patterns. Applications of this system include pharmacological evaluation of the effects of receptor agonists and antagonists on propulsive motility, as well as assessment of changes that result from pathophysiological conditions, such as inflammation or stress. The guinea pig distal colon propulsive motility assay, using the GIMM system, is straightforward and simple to learn, and it provides a reliable and reproducible method of assessing propulsive motility.

Propionate-induced epithelial K+ and Cl−/HCO 3 − secretion and free fatty acid receptor 2 (FFA2, GPR43) expression in the guinea pig distal colon

Propionate, a fermented product in the lumen of the large intestine, is a short-chain fatty acid (SCFA) known to have a variety of localized physiological and pathophysiological functions (e.g., luminal fluid secretion and anti-inflammatory response). In the present study, we investigated propionate-induced transepithelial ion transport and the expression of SCFA receptor, free fatty acid receptor 2 (FFA2, otherwise known as GPR43) in the guinea pig distal colon utilizing the Ussing chamber technique and immunohistochemistry. The addition of propionate to the luminal bathing solution concentration-dependently induced transient K(+) and Cl(-) and/or bicarbonate secretion within approximately 30s and long-lasting Cl(-) secretion for approximately 60min was first identified in the present study. The transient anion secretion was tetrodotoxin (TTX)-sensitive and mediated through the cholinergic (both nicotinic and muscarinic) neural pathway, but the transient K(+) and long-lasting Cl(-) secretion were due to TTX-insensitive mechanism. Immunohistochemistry studies showed that some chromogranin A-immunoreactive enteroendocrine cells were also immunoreactive for FFA2 but not colocalized with 5-hydroxytryptamine. In conclusion, the propionate-induced secretion consisted of the neural and non-neural three-phase secretory manner possibly mediated by the stimulation of FFA2 expressed by enteroendocrine cells.

The traditional antidiarrheal remedy, Garcinia buchananii stem bark extract, inhibits propulsive motility and fast synaptic potentials in the guinea pig distal colon.

Garcinia buchananii bark extract is a traditional African remedy for diarrhea, dysentery, abdominal discomfort, and pain. We investigated the mechanisms and efficacy of this extract using the guinea pig distal colon model of gastrointestinal motility. Stem bark was collected from G. buchananii trees in their natural habitat of Karagwe, Tanzania. Bark was sun dried and ground into fine powder, and suspended in Krebs to obtain an aqueous extract. Isolated guinea pig distal colon was used to determine the effect of the G. buchananii bark extract on fecal pellet propulsion. Intracellular recording was used to evaluate the extract action on evoked fast excitatory postsynaptic potentials (fEPSPs) in S-neurons of the myenteric plexus. Garcinia buchananii bark extract inhibited pellet propulsion in a concentration-dependent manner, with an optimal concentration of ∼10 mg powder per mL Krebs. Interestingly, washout of the extract resulted in an increase in pellet propulsion to a level above basal activity. The extract reversibly reduced the amplitude of evoked fEPSPs in myenteric neurons. The extract's inhibitory action on propulsive motility and fEPSPs was not affected by the opioid receptor antagonist, naloxone, or the alpha- 2 adrenoceptor antagonist, yohimbine. The extract inhibited pellet motility in the presence of gamma-aminobutyric acid (GABA), GABA(A) and GABA(B) receptor antagonists picrotoxin and phaclofen, respectively. However, phaclofen and picrotoxin inhibited recovery rebound of motility during washout. Garcinia buchananii extract has the potential to provide an effective, non-opiate antidiarrheal drug. Further studies are required to characterize bioactive components and elucidate the mechanisms of action, efficacy, and safety.

Enteric Glia Are Targets of the Sympathetic Innervation of the Myenteric Plexus in the Guinea Pig Distal Colon

Astrocytes respond to synaptic activity in the CNS. Astrocytic responses are synapse specific and precisely regulate synaptic activity. Glia in the peripheral nervous system also respond to neuronal activity, but it is unknown whether glial responses are synapse specific. We addressed this issue by examining the activation of enteric glia by distinct neuronal subpopulations in the enteric nervous system. Enteric glia are unique peripheral glia that surround enteric neurons and respond to neuronally released ATP with increases in intracellular calcium ([Ca2+]i). Autonomic control of colonic function is mediated by intrinsic (enteric) and extrinsic (sympathetic, parasympathetic, primary afferent) neural pathways. Here we test the hypothesis that a defined population of neurons activates enteric glia using a variety of techniques to ablate or stimulate components of the autonomic innervation of the colon. Our findings demonstrate that, in the male guinea pig colon, activation of intrinsic neurons does not stimulate glial [Ca2+]i responses and fast enteric neurotransmission is not necessary to initiate glial responses. However, ablating extrinsic innervation significantly reduces glial responses to neuronal activation. Activation of primary afferent fibers does not activate glial [Ca2+]i responses. Selectively ablating sympathetic fibers reduces glial activation to a similar extent as total extrinsic denervation. Neuronal activation of glia follows the same frequency dependence as sympathetic neurotransmitter release, but the only sympathetic neurotransmitter that activates glial [Ca2+]i responses is ATP, suggesting that sympathetic fibers release ATP to activate enteric glia. Therefore, enteric glia discern activity in adjacent synaptic pathways and selectively respond to sympathetic activation.

S1799 Specialized Spinal Afferent Neurons to Detect Mucosal Shear in Guinea-Pig Colon

Background and aims. Extrinsic sensory neurons to the gut mediate both sensation and gastrointestinal reflexes important for gut function in health and disease. Different classes of afferents which convey various types of information about the gut wall and contents can be reliably identified by characterizing the morphology of their physiologically-identified peripheral axons. “Muscular mucosal” afferents have been identified on the basis of their responses to both colorectal distension and to light mucosal stroking. Methods. Extracellular recording from colonic nerves in isolated preparations of guinea pig distal colon were combined with biotinamide filling In Vitro. Results. When all layers of the colon were present in the preparation, a class of afferents could be strongly activated by light von Frey hairs (0.1 10 mN, maximum instantaneous firing: 83+/-18 Hz, n=6). The same units could also be activated by circumferential distension with constant loads (10 100mN) evoking firing of 52+/-30 Hz (n=10). Both types of activity were preserved in preparations where the muscularis externa had been removed prior to recording, suggesting that transduction sites were located in the submucosa or mucosa. Surprisingly, afferents of this class also fired in concert with spontaneous contractions of preparations, measured under isotonic conditions. The action potentials of these units were significantly larger and of shorter duration than action potentials from mechanonociceptors axons recorded from the same nerve trunks (341+/-146 vs 139+/-34μV P<0.01, 0.4+/-0.1 vs 1.1+/-0.8 ms, P<0.05, n=9). None of 9 “muscular mucosal” axons recorded responded to focal application of capsaicin (0.3 μM) and their mechanosensitivity persisted in calcium-free solution (with raised [Mg++]). This suggests that they are intrinsically mechanosensitive rather than relying on release of mediators from other cells. Biotinamide dye filling revealed fine branching axons running in the subepithelial plexus in regions corresponding closely to mechanosensitive receptive fields of these units (n=4). Conclusions. We conclude that “muscular mucosal” afferents comprise a discrete and distinctive class of spinal afferent innervation, well suited to detect physiological levels of mucosal shear in the distal colon of the guinea-pig.

T2056 Purinergic Inhibitory Junction Potentials are Selectively Attenuated in Regions of Disrupted Motility in the Inflamed Guinea Pig Distal Colon

T2056 Purinergic Inhibitory Junction Potentials are Selectively Attenuated in Regions of Disrupted Motility in the Inflamed Guinea Pig Distal Colon