Neural targets of the enteric dopaminergic system in regulating motility of rat proximal colon.

In isolated segments of the rat proximal colon, the dopamine reuptake inhibitor GBR 12909 (GBR) causes a dilatation, while the D1-like receptor antagonist SCH 23390 (SCH) induces a tonic constriction, suggesting that neurally released dopamine tonically stimulates enteric inhibitory efferent neurons. Here, the targets of the enteric dopaminergic neurons were investigated. Cannulated segments of rat proximal colon were bathed in physiological salt solution and luminally perfused with 0.9% saline, while all drugs were applied to the bath. Spatio-temporal maps of colonic motility were constructed from video recordings of peristaltic contractions, and the maximum diameter was measured as an index of colonic contractility. GBR (1 µM)-induced dilatations of colonic segments were prevented by SCH (5 µM), L-nitro arginine (L-NA; 100 µM), a nitric oxide synthase inhibitor, or tetrodotoxin (0.6 µM). In contrast, constrictions induced by a higher concentration of SCH (20 µM) were unaffected by either L-NA or tetrodotoxin. The vasoactive intestinal peptide (VIP) receptor antagonist VIP10-28 (3 µM) or P2Y1 receptor antagonist MRS 2500 (1 µM) had no effect on either the GBR-induced dilatation or the SCH-induced constriction. In colonic segments that had been pretreated with 6-hydroxydopamine (100 µM, 3 h) to deplete enteric dopamine, GBR failed to increase the colonic diameter, while SCH was still capable of constricting colonic segments. Enteric dopaminergic neurons appear to project to nitrergic neurons to dilate the proximal colon by activating neuronal D1-like receptors. In addition, constitutively activated D1-like receptors expressed in cells yet to be determined may provide a tonic inhibition on colonic constrictions.

Mechanisms underlying the prokinetic effects of endogenous glucagon-like peptide-1 in the rat proximal colon.

Glucagon-like peptide-1 (GLP-1), a well-known insulin secretagogue, is released from enteroendocrine L cells both luminally and basolaterally to exert different effects. Basolaterally released GLP-1 increases epithelial ion transport by activating CGRP-containing enteric afferent neurons. Although bath-applied GLP-1 reduced the contractility of colonic segments, GLP-1-induced stimulation of afferent neurons could also accelerate peristaltic contractions. Here, the roles of endogenous GLP-1 in regulating colonic peristalsis were investigated using isolated colonic segments. Isolated segments of rat proximal colon were placed in an organ bath, serosally perfused with oxygenated physiological salt solution, and luminally perfused with degassed 0.9% saline. Colonic wall motion was recorded using a video camera and converted into spatiotemporal maps. Intraluminal administration of GLP-1 (100 nM) stimulating the secretion of GLP-1 from L cells increased the frequency of oro-aboral propagating peristaltic contractions. The acceleratory effect of GLP-1 was blocked by luminally applied exendin-3 (9-39) (100 nM), a GLP-1 receptor antagonist. GLP-1-induced acceleration of peristaltic contractions was also prevented by bath-applied BIBN4069 (1 µM), a CGRP receptor antagonist. In colonic segments that had been exposed to bath-applied capsaicin (100 nM) that desensitizes extrinsic afferents, GLP-1 was still capable of exerting its prokinetic effect. Stimulation of endogenous GLP-1 secretion with a luminally applied cocktail of short-chain fatty acids (1 mM) increased the frequency of peristaltic waves in an exendin-3 (9-39)-sensitive manner. Thus, GLP-1 activates CGRP-expressing intrinsic afferents to accelerate peristalsis in the proximal colon. Short-chain fatty acids appear to stimulate endogenous GLP-1 secretion from L cells resulting in the acceleration of colonic peristalsis.NEW & NOTEWORTHY Glucagon-like peptide-1 (GLP-1) activates CGRP-containing intrinsic afferent neurons resulting in the acceleration of colonic peristalsis. Short-chain fatty acids stimulate the secretion of endogenous GLP-1 from L cells that accelerates colonic peristalsis. Thus, besides the well-known humoral insulinotropic action, GLP-1 exerts a local action via the activation of the enteric nervous system to accelerate colonic motility. Such a prokinetic action of GLP-1 could underlie the mechanisms causing diarrhea in patients with type-2 diabetes treated with GLP-1 analogs.

Role of enteric dopaminergic neurons in regulating peristalsis of rat proximal colon.

BACKGROUND: Constipation is commonly seen in patients with Parkinson's disease associated with a loss of dopaminergic neurons in both central and enteric nervous systems. However, the roles of enteric dopaminergic neurons in developing constipation remain to be elucidated. Here, we investigated the roles of enteric dopaminergic neurons in the generation of colonic peristalsis. METHODS: Cannulated segments of rat proximal colon were situated in the organ bath, abluminally perfused with physiological salt solution and luminally perfused with 0.9% saline. Drugs were applied in the abluminal solution. Changes in diameter along the length of the colonic segment were captured by a video camera and transformed into spatio-temporal maps. Fluorescence immunohistochemistry was also carried out. KEY RESULTS: Blockade of nitrergic neurotransmission prevented oro-aboral propagation of peristaltic waves and caused a colonic constriction without affecting ripples, non-propagating myogenic contractions. Blockade of cholinergic neurotransmission also prevented peristaltic waves but suppressed ripples with a colonic dilatation. Tetrodotoxin (0.6 µM) abolished peristaltic waves and increased ripples with a constriction. SCH 23390 (20 µM), a D1 -like dopamine receptor antagonist, slowed the peristaltic waves and caused a constriction, while GBR 12909 (1 µM), a dopamine reuptake inhibitor, diminished the peristaltic waves with a dilatation. Bath-applied dopamine (3 µM) abolished the peristaltic waves associated with a colonic dilation in an SCH 23390 (5 µM)-sensitive manner. D1 receptor immunoreactivity was co-localized to nitrergic and cholinergic neurons. CONCLUSIONS AND INFERENCES: Dopaminergic neurons appear to facilitate nitrergic neurons via D1 -like receptors to stabilize asynchronous contractile activity resulting in the generation of colonic peristalsis.

α-MSH-induced activation of spinal MC1R but not MC4R enhances colorectal motility in anaesthetised rats.

The central nervous system is involved in regulation of defaecation. It is generally considered that supraspinal regions control the spinal defaecation centre. However, signal transmission from supraspinal regions to the spinal defaecation centre is still unclear. In this study, we investigated the regulatory role of an anorexigenic neuropeptide, α-MSH, in the spinal defaecation centre in rats. Intrathecal administration of α-MSH to the L6-S1 spinal cord enhanced colorectal motility. The prokinetic effect of α-MSH was abolished by severing the pelvic nerves. In contrast, severing the colonic nerves or thoracic cord transection at the T4 level had no impact on the effect of α-MSH. RT-PCR analysis revealed MC1R mRNA and MC4R mRNA expression in the L6-S1 spinal cord. Intrathecally administered MC1R agonists, BMS470539 and SHU9119, mimicked the α-MSH effect, but a MC4R agonist, THIQ, had no effect. These results demonstrate that α-MSH binds to MC1R in the spinal defaecation centre and activates pelvic nerves, leading to enhancement of colorectal motility. This is, to our knowledge, the first report showing the functional role of α-MSH in the spinal cord. In conclusion, our findings suggest that α-MSH is a candidate for a neurotransmitter from supraspinal regions to the spinal defaecation centre.

Sexually dimorphic response of colorectal motility to noxious stimuli in the colorectum in rats.

KEY POINTS: This study showed a remarkable sex difference in responses of colorectal motility to noxious stimuli in the colorectum in rats: colorectal motility was enhanced in response to intracolonic administration of a noxious stimulant, capsaicin, in male rats but not in female rats. The difference in descending neurons from the brain to spinal cord operating after noxious stimulation could be responsible for the sex difference. In male rats, serotoninergic and dopaminergic neurons are dominantly activated, both of which activate the spinal defaecation centre. In female rats, GABAergic neurons in addition to serotoninergic neurons are activated. GABA may compete for facilitative action of 5-HT in the spinal defaecation centre, and thereby colorectal motility is not enhanced in response to intracolonic administration of capsaicin. The findings provide a novel insight into pathophysiological mechanisms of sex differences in functional defaecation disorders such as irritable bowel syndrome. ABSTRACT: We previously demonstrated that noxious stimuli in the colorectum enhance colorectal motility through activation of descending pain inhibitory pathways in male rats. It can be expected that the regulatory mechanisms of colorectal motility differ in males and females owing to remarkable sex differences in descending pain inhibitory pathways. Thus, we aimed to clarify sex differences in responses of colorectal motility to noxious stimuli in rats. Colorectal motility was measured in vivo in anaesthetized rats. Administration of a noxious stimulant, capsaicin, into the colorectal lumen enhanced colorectal motility in male rats but not in female rats. Quantitative PCR and immunohistochemistry showed that TRPV1 expression levels in the dorsal root ganglia and in the colorectal mucosa were comparable in male and female rats. When a GABAA receptor inhibitor was intrathecally administered to the L6-S1 level of the spinal cord, colorectal motility was facilitated in response to intracolonic capsaicin even in female rats. The capsaicin-induced response in the presence of the GABA blocker in female rats was inhibited by intrathecal administration of 5-HT2 and -3 receptor antagonists but not by a D2-like dopamine receptor antagonist. Our findings demonstrate that intracolonic noxious stimulation activates GABAergic and serotoninergic descending neurons in female rats, whereas serotoninergic and dopaminergic neurons are dominantly activated in male rats. Thus, the difference in the descending neurons operating after noxious stimulation would be responsible for the sexually dimorphic responses of colorectal motility. Our findings provide a novel insight into pathophysiological mechanisms of sex differences in functional defaecation disorders such as irritable bowel syndrome.

Roles of the noradrenergic nucleus locus coeruleus and dopaminergic nucleus A11 region as supraspinal defecation centers in rats.

We previously demonstrated that administration of norepinephrine, dopamine, and serotonin into the lumbosacral defecation center caused propulsive contractions of the colorectum. It is known that the monoamines in the spinal cord are released mainly from descending neurons in the brainstem. In fact, stimulation of the medullary raphe nuclei, the origin of descending serotonergic neurons, enhances colorectal motility via the lumbosacral defecation center. Therefore, the purpose of this study was to examine the roles of the noradrenergic nucleus locus coeruleus (LC) and dopaminergic nucleus A11 region in the defecation reflex. Colorectal motility was measured with a balloon in anesthetized rats. Electrical stimulation of the LC and A11 region increased colorectal pressure only when a GABAA receptor antagonist was injected into the lumbosacral spinal cord. The effects of the LC stimulation and A11 region stimulation on colorectal motility were inhibited by antagonists of α1-adrenoceptors and D2-like dopamine receptors injected into the lumbosacral spinal cord, respectively. Spinal injection of a norepinephrine-dopamine reuptake inhibitor augmented the colokinetic effect of LC stimulation. The effect of stimulation of each nucleus was abolished by surgical severing of the parasympathetic pelvic nerves. Our findings demonstrate that activation of descending noradrenergic neurons from the LC and descending dopaminergic neurons from the A11 region causes enhancement of colorectal motility via the lumbosacral defecation center. The present study provides a novel concept that the brainstem monoaminergic nuclei play a role as supraspinal defecation centers.NEW & NOTEWORTHY The present study demonstrates that electrical and chemical stimulations of the locus coeruleus or A11 region augment contractions of the colorectum. The effects of locus coeruleus and A11 stimulations on colorectal motility are due to activation of α1-adrenoceptors and D2-like dopamine receptors in the lumbosacral defecation center, respectively. The present study provides a novel concept that the brainstem monoaminergic nuclei play a role as supraspinal defecation centers.

Characterization of peristaltic motility in the striated muscle portion of the esophagus using a novel in vivo method in rats.

BACKGROUND: Esophageal peristalsis is controlled by the brainstem via vago-vagal reflex. However, the precise regulatory mechanisms in the striated muscle portion are largely unknown. The aim of this study was to characterize peristaltic motility in the portion of the esophagus using a novel in vivo method in rats. METHODS: A balloon-tipped catheter was placed in the esophagus of a rat anesthetized with urethane. To induce esophageal peristalsis, the balloon was inflated by water injection. KEY RESULTS: When the balloon was inflated near the bronchial bifurcation, the balloon was transported in the aboral direction. Vagotomy abolished the peristaltic response. The threshold volume for inducing esophageal peristalsis varied according to the velocity of balloon distention; the volume being effective to induce peristalsis at a low inflation speed was smaller than the threshold volume at a rapid inflation speed. Even in the absence of inflation, keeping the balloon inside the esophagus during an interval period prevented subsequent induction of peristaltic motility. In addition, a nitric oxide synthase inhibitor abolished the induction of esophageal peristalsis. CONCLUSIONS AND INFERENCES: Our findings suggest that (a) in addition to the intensity, the velocity of distention is important for activating the mechanosensory mechanism to induce esophageal peristalsis, (b) tonic inputs from afferent fibers located at the mucosa may reduce the excitability of mechanosensors which is necessary for inducing peristalsis, and (c) nitric oxide plays essential roles in the induction of esophageal peristalsis. These results provide novel insights into the regulatory mechanisms of esophageal motility.

Descending monoaminergic pathways projecting to the spinal defecation center enhance colorectal motility in rats.

The central regulating mechanisms of defecation, especially roles of the spinal defecation center, are still unclear. We have shown that monoamines including norepinephrine, dopamine, and serotonin injected into the spinal defecation center cause propulsive contractions of the colorectum. These monoamines are the main neurotransmitters of descending pain inhibitory pathways. Therefore, we hypothesized that noxious stimuli in the colorectum would activate the descending monoaminergic pathways projecting to the spinal defecation center and that subsequently released endogenous monoamine neurotransmitters would enhance colorectal motility. Colorectal motility was measured in rats anesthetized with α-chloralose and ketamine. As a noxious stimulus, capsaicin was administered into the colorectal lumen. To interrupt neuronal transmission in the spinal defecation center, antagonists of norepinephrine, dopamine, and/or serotonin receptors were injected intrathecally at the L6-S1 spinal level, where the spinal defecation center is located. Intraluminal administration of capsaicin, acting on the transient receptor potential vanilloid 1 channel, caused transient propulsive contractions. The effect of capsaicin was abolished by surgical severing of the pelvic nerves or thoracic spinal transection at the T4 level. Capsaicin-induced contractions were blocked by preinjection of D2-like dopamine receptor and 5-hydroxytryptamine subtype 2 and 3 receptor antagonists into the spinal defecation center. We demonstrated that intraluminally administered capsaicin causes propulsive colorectal motility through reflex pathways involving the spinal and supraspinal defecation centers. Our results provide evidence that descending monoaminergic neurons are activated by noxious stimulation to the colorectum, leading to facilitation of colorectal motility. NEW & NOTEWORTHY The present study demonstrates that noxious stimuli in the colorectum activates the descending monoaminergic pathways projecting to the spinal defecation center and that subsequently released endogenous monoamine neurotransmitters, serotonin and dopamine, enhance colorectal motility. Our findings provide a possible explanation of the concurrent appearance of abdominal pain and bowel disorder in irritable bowel syndrome patients. Thus the present study may provide new insights into understanding of mechanisms of colorectal dysfunction involving the central nervous system.

Local regulatory mechanism to coordinate colorectal motility in rats.

The presence of a fecal pellet in the colorectum causes ascending contraction and descending relaxation, propelling the pellet aborally. However, random occurrence of the reflexes at multiple sites would disturb sequential excretion of the pellets, resulting in inefficient defecation. Hence, we postulated that a regulatory mechanism to coordinate peristaltic motility initiated at adjacent portions of the colorectum may exist. Colorectal motility was recorded with balloons located at 2 cm, 5 cm and 7 cm from the anus in vivo in anesthetized rats. The presence of a balloon in the colorectum inhibited motility of the oral side and enhanced motility of the anal side. Both the ascending inhibitory and descending facilitatory actions were unaffected by cutting the pelvic nerves, suggesting little contribution of the lumbosacral defecation center. In contrast, disrupting the continuity of the enteric nervous system abolished the local reflex mechanism. The ascending inhibitory pathway operated in a condition in which facilitatory input from the lumbosacral defecation center was fully activated by intrathecal injection of ghrelin. We also found that functional impairment of the local reflex pathways was evident in rats that recovered from 2,4,6-trinitrobenzensulfonic acid-induced colitis. These results demonstrate that an intrinsic regulatory mechanism to coordinate peristaltic motility initiated at adjacent portions exists in the rat colorectum. The regulation may be beneficial to propel multiple pellets efficiently. In addition, impairment of the local regulatory mechanism might be involved in postinflammatory dysmotility in the colorectum.

Colokinetic effect of somatostatin in the spinal defecation center in rats.

Somatostatin and its receptors are expressed in the spinal cord, but the functional roles of the peptide remain unknown. In this study, we examined the colokinetic effect of somatostatin in the spinal defecation center in anesthetized rats. Intrathecal application of somatostatin into the lumbo-sacral cord caused propulsive contractions of the colorectum. However, somatostatin administered intravenously or intrathecally to the thoracic cord failed to enhance colorectal motility. Transection of the thoracic cord had no significant impact on the colokinetic action of somatostatin. The enhancement of colorectal motility by intrathecal administration of somatostatin was abolished by severing the pelvic nerves. Our results demonstrate that somatostatin acting on the spinal defecation center causes propulsive motility of the colorectum in rats. Considering that somatostatin is involved in nociceptive signal transmission in the spinal cord, our results provide a rational explanation for the concurrent appearance of chronic abdominal pain and colonic motility disorders in IBS patients.

Medullary raphe nuclei activate the lumbosacral defecation center through the descending serotonergic pathway to regulate colorectal motility in rats.

Colorectal motility is regulated by two defecation centers located in the brain and spinal cord. In previous studies, we have shown that administration of serotonin (5-HT) in the lumbosacral spinal cord causes enhancement of colorectal motility. Because spinal 5-HT is derived from neurons of the medullary raphe nuclei, including the raphe magnus, raphe obscurus, and raphe pallidus, we examined whether stimulation of the medullary raphe nuclei enhances colorectal motility via the lumbosacral defecation center. Colorectal pressure was recorded with a balloon in vivo in anesthetized rats. Electrical stimulation of the medullary raphe nuclei failed to enhance colorectal motility. Because GABAergic neurons can be simultaneously activated by the raphe stimulation and released GABA masks accelerating actions of the raphe nuclei on the lumbosacral defecation center, a GABAA receptor antagonist was preinjected intrathecally to manifest excitatory responses. When spinal GABAA receptors were blocked by the antagonist, electrical stimulation of the medullary raphe nuclei increased colorectal contractions. This effect of the raphe nuclei was inhibited by intrathecal injection of 5-hydroxytryptamine type 2 (5-HT2) and type 3 (5-HT3) receptor antagonists. In addition, injection of a selective 5-HT reuptake inhibitor in the lumbosacral spinal cord augmented the raphe stimulation-induced enhancement of colorectal motility. Transection of the pelvic nerves, but not transection of the colonic nerves, prevented the effect of the raphe nuclei on colorectal motility. These results demonstrate that activation of the medullary raphe nuclei causes augmented contractions of the colorectum via 5-HT2 and 5-HT3 receptors in the lumbosacral defecation center. NEW & NOTEWORTHY We have shown that electrical stimulation of the medullary raphe nuclei causes augmented contractions of the colorectum via pelvic nerves in rats. The effect of the medullary raphe nuclei on colorectal motility is exerted through activation of 5-hydroxytryptamine type 2 and type 3 receptors in the lumbosacral defecation center. The descending serotoninergic raphespinal tract represents new potential therapeutic targets against colorectal dysmotility such as irritable bowel syndrome.

Induction of hibernation-like hypothermia by central activation of the A1 adenosine receptor in a non-hibernator, the rat.

Central adenosine A1-receptor (A1AR)-mediated signals play a role in the induction of hibernation. We determined whether activation of the central A1AR enables rats to maintain normal sinus rhythm even after their body temperature has decreased to less than 20 °C. Intracerebroventricular injection of an adenosine A1 agonist, N6-cyclohexyladenosine (CHA), followed by cooling decreased the body temperature of rats to less than 20 °C. Normal sinus rhythm was fundamentally maintained during the extreme hypothermia. In contrast, forced induction of hypothermia by cooling anesthetized rats caused cardiac arrest. Additional administration of pentobarbital to rats in which hypothermia was induced by CHA also caused cardiac arrest, suggesting that the operation of some beneficial mechanisms that are not activated under anesthesia may be essential to keep heart beat under the hypothermia. These results suggest that central A1AR-mediated signals in the absence of anesthetics would provide an appropriate condition for maintaining normal sinus rhythm during extreme hypothermia.

Stimulation of dopamine D2-like receptors in the lumbosacral defaecation centre causes propulsive colorectal contractions in rats.

KEY POINTS: The pathophysiological roles of the CNS in bowel dysfunction in patients with irritable bowel syndrome and Parkinson's disease remain obscure. In the present study, we demonstrate that dopamine in the lumbosacral defaecation centre causes strong propulsive motility of the colorectum. The effect of dopamine is a result of activation of sacral parasympathetic preganglionic neurons via D2-like dopamine receptors. Considering that dopamine is a neurotransmitter of descending pain inhibitory pathways, our results highlight the novel concept that descending pain inhibitory pathways control not only pain, but also the defaecation reflex. In addition, severe constipation in patients with Parkinson's disease can be explained by reduced parasympathetic outflow as a result of a loss of the effect of dopaminergic neurons. ABSTRACT: We have recently demonstrated that intrathecally injected noradrenaline caused propulsive contractions of the colorectum. We hypothesized that descending pain inhibitory pathways control not only pain, but also the defaecation reflex. Because dopamine is one of the major neurotransmitters of descending pain inhibitory pathways in the spinal cord, we examined the effects of the intrathecal application of dopamine to the spinal defaecation centre on colorectal motility. Colorectal intraluminal pressure and expelled volume were recorded in vivo in anaesthetized rats. Slice patch clamp and immunohistochemistry were used to confirm the existence of dopamine-sensitive neurons in the sacral parasympathetic nuclei. Intrathecal application of dopamine into the L6-S1 spinal cord, where the lumbosacral defaecation centre is located, caused propulsive contractions of the colorectum. Inactivation of spinal neurons using TTX blocked the effect of dopamine. Although thoracic spinal transection had no effect on the enhancement of colorectal motility by intrathecal dopamine, the severing of the pelvic nerves abolished the enhanced motility. Pharmacological experiments revealed that the effect of dopamine is mediated primarily by D2-like dopamine receptors. Neurons labelled with retrograde dye injected at the colorectum showed an inward current in response to dopamine in slice patch clamp recordings. Furthermore, immunohistochemical analysis revealed that neurons immunoreactive to choline acetyltransferase express D2-like dopamine receptors. Taken together, our findings demonstrate that dopamine activates sacral parasympathetic preganglionic neurons via D2-like dopamine receptors and causes propulsive motility of the colorectum in rats. The present study supports the hypothesis that descending pain inhibitory pathways regulate defaecation reflexes.

Does the capsaicin-sensitive local neural circuit constitutively regulate vagally evoked esophageal striated muscle contraction in rats?

To determine whether a capsaicin-sensitive local neural circuit constitutively modulates vagal neuromuscular transmission in the esophageal striated muscle or whether the neural circuit operates in a stimulus-dependent manner, we compared the motility of esophageal preparations isolated from intact rats with those in which capsaicin-sensitive neurons had been destroyed. Electrical stimulation of the vagus nerve trunk evoked contractile responses in the esophagus isolated from a capsaicin-treated rat in a manner similar to those in the esophagus from a control rat. No obvious differences were observed in the inhibitory effects of D-tubocurarine on intact and capsaicin-treated rat esophageal motility. Destruction of the capsaicin-sensitive neurons did not significantly affect latency, time to peak and duration of a vagally evoked twitch-like contraction. These findings indicate that the capsaicin-sensitive neural circuit does not operate constitutively but rather is activated in response to an applied stimulus.

Inhibitory action of hydrogen sulfide on esophageal striated muscle motility in rats.

Hydrogen sulfide (H2S) is recognized as a gaseous transmitter and has many functions including regulation of gastrointestinal motility. The aim of the present study was to clarify the effects of H2S on the motility of esophageal striated muscle in rats. An isolated segment of the rat esophagus was placed in an organ bath and mechanical responses were recorded using a force transducer. Electrical stimulation of the vagus nerve evoked contractile response in the esophageal segment. The vagally mediated contraction was inhibited by application of an H2S donor. The H2S donor did not affect the contraction induced by electrical field stimulation, which can excite the striated muscle directly, not via vagus nerves. These results show that H2S has an inhibitory effect on esophageal motility not by directly attenuating striated muscle contractility but by blocking vagal motor nerve activity and/or neuromuscular transmissions. The inhibitory actions of H2S were not affected by pretreatment with the transient receptor potential vanniloid-1 blocker, transient receptor potential ankyrin-1 blocker, nitric oxide synthase inhibitor, blockers of potassium channels, and ganglionic blocker. RT-PCR and Western blot analysis revealed the expression of H2S-producing enzymes in esophageal tissue, whereas application of inhibitors of H2S-producing enzymes did not change vagally evoked contractions in the esophageal striated muscle. These findings suggest that H2S, which might be produced in the esophageal tissue endogenously, can regulate the motor activity of esophageal striated muscle via a novel inhibitory neural pathway.

Actions of probiotics on trinitrobenzenesulfonic acid-induced colitis in rats.

We investigated the actions of probiotics, Streptococcus faecalis 129 BIO 3B (SF3B), in a trinitrobenzenesulfonic acid- (TNBS-) induced colitis model in rats. After TNBS was administered into the colons of rats for induction of colitis, the rats were divided into two groups: one group was given a control diet and the other group was given a diet containing SF3B for 14 days. There were no apparent differences in body weight, diarrhea period, macroscopic colitis score, and colonic weight/length ratio between the control group and SF3B group, suggesting that induction of colitis was not prevented by SF3B. Next, we investigated whether SF3B-containing diet intake affects the restoration of enteric neurotransmissions being damaged during induction of colitis by TNBS using isolated colonic preparations. Recovery of the nitrergic component was greater in the SF3B group than in the control group. A compensatory appearance of nontachykininergic and noncholinergic excitatory components was less in the SF3B group than in the control group. In conclusion, the present study suggests that SF3B-containing diet intake can partially prevent disruptions of enteric neurotransmissions induced after onset of TNBS-induced colitis, suggesting that SF3B has therapeutic potential.

Colokinetic effect of noradrenaline in the spinal defecation center: implication for motility disorders.

Chronic abdominal pain in irritable bowel syndrome (IBS) usually appears in combination with disturbed bowel habits, but the etiological relationship between these symptoms remains unclear. Noradrenaline is a major neurotransmitter controlling pain sensation in the spinal cord. To test the hypothesis that the descending noradrenergic pathway from the brain stem moderates gut motility, we examined effects of intrathecal application of noradrenaline to the spinal defecation center on colorectal motility. Colorectal intraluminal pressure and expelled volume were recorded in vivo in anesthetized rats. Intrathecal application of noradrenaline into the L6-S1 spinal cord, where the lumbosacral defecation center is located, caused propulsive contractions of the colorectum. Inactivation of spinal neurons by tetrodotoxin blocked the effect of noradrenaline. Pharmacological experiments showed that the effect of noradrenaline is mediated primarily by alpha-1 adrenoceptors. The enhancement of colorectal motility by intrathecal noradrenaline was abolished by severing of the pelvic nerves. Our results demonstrate that noradrenaline acting on sacral parasympathetic preganglionic neurons through alpha-1 adrenoceptors causes propulsive motility of the colorectum in rats. Considering that visceral pain activates the descending inhibitory pathways including noradrenergic neurons, our results provide a rational explanation of the concurrent appearance of chronic abdominal pain and colonic motility disorders in IBS patients.

Hibernation-specific alternative splicing of the mRNA encoding cold-inducible RNA-binding protein in the hearts of hamsters.

The hearts of hibernating animals are capable of maintaining constant beating despite a decrease in body temperature to less than 10 °C during hibernation, suggesting that the hearts of hibernators are highly tolerant to a cold temperature. In the present study, we examined the expression pattern of cold-inducible RNA-binding protein (CIRP) in the hearts of hibernating hamsters, since CIRP plays important roles in protection of various types of cells against harmful effects of cold temperature. RT-PCR analysis revealed that CIRP mRNA is constitutively expressed in the heart of a non-hibernating euthermic hamster with several different forms probably due to alternative splicing. The short product contained the complete open reading frame for full-length CIRP. On the other hand, the long product had inserted sequences containing a stop codon, suggesting production of a C-terminal deletion isoform of CIRP. In contrast to non-hibernating hamsters, only the short product was amplified in hibernating animals. Induction of artificial hypothermia in non-hibernating hamsters did not completely mimic the splicing patterns observed in hibernating animals, although a partial shift from long form mRNA to short form was observed. Our results indicate that CIRP expression in the hamster heart is regulated at the level of alternative splicing, which would permit a rapid increment of functional CIRP when entering hibernation.

Regulation of longitudinal esophageal motility in the house musk shrew (Suncus murinus).

Suncus murinus (house musk shrew; suncus) is a species of insectivore that has an ability to vomit. Although longitudinal movement of the esophagus would be related to the emetic response, regulatory mechanisms for the suncus esophageal motility are unclear. Therefore, the aim of the present study was to clarify components that regulate esophageal motility in the suncus. An isolated segment of the suncus esophagus was placed in an organ bath, and longitudinal mechanical responses were recorded using a force transducer. Electrical stimulation of the vagus trunk evoked a biphasic contractile response. The first phase of the contractile response was blocked by α-bungarotoxin, a blocker of nicotinic acetylcholine receptors on striated muscle cells, whereas the second one was blocked by atropine, a blocker of muscarinic acetylcholine receptors on smooth muscle cells. Next, we investigated whether mast cells are involved in motor functions of the suncus esophagus. Application of a mast cell stimulator, compound 48/80, elicited contractile responses, which was resistant to tetrodotoxin. Exogenous application of serotonin and histamine induced contractile responses. The mast cell activation-mediated contraction was abolished by double desensitization by serotonin and histamine and pre-treatment with indomethacin, a cyclooxygenase inhibitor. The findings show that cholinergic and non-cholinergic transmitters induce longitudinal contraction in the suncus esophagus, which might contribute to esophageal shortening during emesis. Cholinergic transmitters are derived from vagal efferents, and non-cholinergic transmitters, which are thought to be serotonin, histamine and prostaglandins, are released from mast cells.

Functional roles of capsaicin-sensitive intrinsic neural circuit in the regulation of esophageal peristalsis in rats: in vivo studies using a novel method.

A well-developed myenteric plexus exists in the esophagus composed of striated muscle layers, but its functional role in controlling peristaltic movements remains to be clarified. The purpose of this study was to clarify the role of a local neural reflex consisting of capsaicin-sensitive primary afferent neurons and intrinsic neurons in esophageal peristalsis. We firstly devised a method to measure peristaltic movement of esophagus in vivo in rats. Rats were anesthetized with urethane, and esophageal intraluminal pressure and propelled intraluminal liquid volume were recorded. In the experimental system, an intraluminal pressure stimulus evoked periodic changes in intraluminal pressure of the esophagus, which were consistently accompanied by intraluminal liquid propulsion. Bilateral vagotomy abolished changes in intraluminal pressure as well as liquid propulsion. These results indicate that the novel method is appropriate for inducing peristalsis in the esophagus composed of striated muscles. Then, by using the method, we examined functional roles of the local reflex in esophageal peristalsis. For that purpose, we used rats in which capsaicin-sensitive neurons had been destroyed. The esophagus of capsaicin-treated rats showed a multiphasic rise in intraluminal pressure, which may due to noncoordinated contractions of esophageal muscles, whereas a monophasic response was observed in the intact rat esophagus. In addition, destruction of capsaicin-sensitive neurons increased the propelled liquid volume and lowered the pressure threshold for initiating peristalsis. These results suggest that the local neural reflex consisting of capsaicin-sensitive neurons and intrinsic neurons contributes to coordination of peristalsis and suppresses mechanosensory function of vagal afferents in the esophagus.