M3 muscarinic receptor-deficient mice retain bethanechol-mediated intestinal ion transport and are more sensitive to colitis

Acetylcholine (ACh) is an important regulator of intestinal epithelial ion transport via muscarinic or nicotinic ACh receptors. Previous studies emphasize the role of the M3 muscarinic receptor subtype in mediating the effects of cholinergic agonists on intestinal ion transport. With the prevalence of mouse models to study intestinal (patho)physiology, it is crucial that ion transport be understood in this species. Using M3 receptor-deficient (KO) mice and wild-type (WT) mice, we examined M3 receptor contributions to ion transport as well as its role in colitis induced by dextran sodium sulphate (DSS). In the Ussing chambers, ileal and colonic tissue from M3 KO and WT mice displayed similar baseline ion transport properties. Short-circuit current (ISC) responses to the muscarinic receptor agonist bethanechol were slightly decreased in ileal tissue from M3 KO mice compared with tissue from WT mice, whereas responses were not significantly different in colonic tissue. ISC responses to bethanechol were partially inhibited by pirenzepine in WT ileum, but not tetrodotoxin, suggesting involvement of a non-neuronal M1 muscarinic receptor. In the ileum, the M3 receptor may inhibit neuronally evoked ion transport, as indicated by the increased ISC responses to electrical stimulation in tissue from M3 KO mice. Furthermore, whereas all DSS-treated mice developed colitis, M3 KO mice displayed more rapid mass loss and more severe disease than DSS-treated WT mice, even following a reduction in the amount and time of DSS treatment. Thus, M3 receptor-KO mice are compensated in their ability to evoke muscarinic receptor-driven ion transport responses, but are more sensitive to DSS. This work highlights the need to dissect muscarinic receptor-mediated events in the mouse, as mice become increasingly valuable in enteric disease models.     

Neural control of intestinal ion transport and paracellular permeability is altered by nutritional status

This study examined the effect of fasting on the neural control of ion transport and paracellular permeability in piglet jejunum. Muscle-stripped tissues from fed or 48-h fasted piglets were mounted in Ussing chambers. Neural blockade with tetrodotoxin (TTX) or antagonists of muscarinic or nicotinic receptors caused reductions in basal short-circuit current that were approximately threefold greater in fasted piglets. The TTX-induced reduction in short-circuit current in fasted piglets was due to a decrease in residual ion flux and was abolished in the absence of HCO(-)(3). Intestinal paracellular permeability, as indicated by tissue conductance (G(t)) and fluxes of inulin and mannitol, was significantly increased by fasting. TTX increased inulin flux and G(t) in fed but not fasted piglets. In fasted piglets, carbachol reduced G(t) by 29% and mannitol flux by 27% but had no effect on these parameters in the fed state. We conclude that fasting enhances enteric neural control of basal ion transport and increases paracellular permeability in piglet jejunum. Tonic release of enteric neurotransmitters regulates paracellular permeability in the fed state, and cholinergic stimulation restores fasting-induced elevations in paracellular permeability to fed levels.     

Actions of a nicotinic agonist,DMPP, on intestinal ion transport invitro

High-dose carbachol (10^?3 M) has previously been shown to cause NaCl absorption in short-circuited rabbit ileum. The mechanism of this effect may be norepinephrine release induced by carbachol activation of presynaptic nicotinic receptors on adrenergic neurons. Norepinephrine then interacts with postsynaptic α-adrenergic receptors on intestinal mucosal cells to stimulate neutral NaCl absorption and inhibit electrogenic bicarbonate secretion. The present paper examines the $\text{in vitro}$ intestinal ion transport effects of DMPP an agent which is more specific than carbachol on nicotinic cholinergic receptors. DMPP (10^?5 M) caused a transient increase followed by prolonged depression of the short-circuit current, increased NaCl absorption and increased tissue conductance. This effect was antagonized by hexamethonium and phentolamine. It is concluded that nicotinic cholinergic agents stimulate norepinephrine release from adrenergic nerves and effect intestinal ion transport just as norepinephrine does.     

Acetylarsenocholine: A Cholinergic Agonist

Abstract: The pharmacological properties of acetylarsenocholine, an arsenic analogue of acetylcholine, were investigated. Acetylarsenocholine behaved as a cholinergic ligand both in the central and peripheral nervous system. It bound to nicotinic receptors in rat medulla-pons with a K _D of 15 μ M and to muscarinic receptors in rat cerebral cortex with a K _D of 10 μ M . It behaved also as an agonist at presynaptic muscarinic receptors in guinea pig ileum myenteric plexus preparation. Arsenocholine is an alternative substrate for choline acetyltransferase and acetylarsenocholine is an alternative substrate for acetylcholinesterase.     

Evidence for substance P as a functional neurotransmitter in guinea pig small intestinal mucosa

We showed previously that electrical transmural stimulation (TS) of guinea pig jejunal mucosa in vitro released neurotransmitters from submucosal plexus neurons which caused alterations in ion transport. The present studies were performed to obtain information regarding the identity of the neurotransmitters. The addition of exogenous substance P (SP) to the serosal side of the tissue caused a transient increase in short-circuit current (Isc) which closely mimicked the response to TS. Both TS and SP caused net secretion of Cl- ions by stimulating movement toward the luminal side. Tetrodotoxin abolished the response to TS, inhibited approximately 70% of the response to SP but did not affect the response to urecholine, a cholinergic muscarinic agonist. In the presence of the muscarinic antagonist, atropine, Isc responses to both TS and SP were reduced suggesting that a portion of both responses was due to action on enteric nerves causing release of acetylcholine. Following desensitization of the tissue with supramaximal doses of SP the response to TS was significantly reduced but the response to urecholine was unchanged. In the presence of atropine, SP desensitization reduced the nerve-stimulated response by approximately 65%; treatment of tissue with SP antibodies reduced the response by approximately 55%. Under the same conditions Isc responses to histamine were unaltered. Our results suggest that both SP (or a structurally analogous neurotransmitter) and acetylcholine as well as additional unidentified neurotransmitter(s) are functionally important in the regulation of intestinal ion transport in guinea pig jejunum.     

Actions of gastrin-releasing peptide and related mammalian and amphibian peptides on ion transport in the porcine proximal jejunum

The 27-amino acid peptide gastrin-releasing peptide (GRP) and the decapeptide neuromedin B (NMB) are structurally related to bombesin (BB) and exist within the mammalian small intestine. We examined the actions of porcine GRP and NMB on ion transport in the porcine proximal jejunum in vitro and compared their activities to those of their respective C-terminal amphibian homologs BB and ranatensin (RT). The 4 peptides transiently increased potential difference and short-circuit current (Isc) in jejunal mucosal sheets after their serosal administration in subnanomolar concentrations with an order of potency: GRP approximately RT greater than or equal to NMB greater than BB. BB and RT were more effective in elevating Isc than GRP and NMB; all peptides had variable effects on tissue conductance. Mucosal Isc responses to GRP (1 nM) were due in part to a stimulation of net Cl- secretion. GRP-induced Isc increases were halved by serosal furosemide (0.3 mM) and reduced by 65% and 90% in tissue bathing solutions lacking Cl- or Cl- and HCO3-, respectively. Tetrodotoxin reduced Isc responses to the peptide by 40%; GRP activity remained unaffected after blockade of gut muscarinic or nicotinic cholinergic receptors by atropine or hexamethonium, respectively. These results suggest that GRP and its natural homologs stimulate active electrogenic Cl- secretion in the porcine jejunum through interactions with GRP receptors located in the intestinal mucosa and submucosa.     

Enkephalin regulation of L-valine transport in rabbit ileum

We studied the influence on ionic basal transport (Na+ and Cl-) and L-valine transport of two enkephalins which are not metabolized and act in delta and mu receptors respectively. Transports have been indirectly determined measuring the transepithelial electric potential and the short circuit current. DADLE does not significantly influence ion and amino-acid transport, while DAGO alters both of them in the presence of the myenteric plexus (muscle layers present) or inhibits only L-valine transport in the absence of the plexus (muscle layers removed).     

Morphological and Functional Alterations of the Myenteric Plexus in Rats with TNBS-Induced Colitis

The 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced model of experimental colitis was used to investigate the time-course of alterations in enteric neurotransmission and/or smooth muscle function that occur in chronic inflammation. Myenteric plexus morphology (immunocytochemical markers), functional integrity of cholinergic neurons (³H-choline uptake, acetylcholine release and contractile response to electrical field stimulation) and smooth muscle integrity (contractile response to exogenous acetylcholine) were determined 2, 7, 15, and 30 days after TNBS treatment. In TNBS-treated rats extensive ulcerations of the mucosa and/or the submucosa and increase in colonic weights were accompanied by significant reduction in ³H-choline uptake, acetylcholine release and contractile response to stimulation of enteric nerves. These changes were maximal 7 and 15 days after TNBS treatment. Immunocytochemical marker (PGP 9.5, SNAP 25, synaptophysin and S100 protein) expression was absent in necrotic areas of colons removed 7 days post-injury and partially reduced in colons removed 15 days after TNBS treatment. By contrast, the contractile response to exogenous acetylcholine was significantly increased after 7 days in both inflamed and uninflamed regions and returned to control values by day 30. Likewise, an almost complete recovery of neural cholinergic function and of myenteric plexus morphology was observed 30 days after TNBS treatment. These data suggest that TNBS-induced colitis is associated with progressive and selective alterations in myenteric plexus structure and function, with consequent reduction of cholinergic neurotransmission and abnormality in colonic contractility. The reversibility of myenteric plexus disruption is a clear indication of neuronal plasticity within enteric nervous system as an adaptative mechanism against inflammatory challenges.     

The effect of leucine-enkephalin on the peristaltic reflex of the isolated guinea-pig ileum

Leucine (leu)-enkephalin depresses or inhibits the peristaltic reflex of the isolated guinea-pig ileum. Opiate antagonists (naloxone and nalorphine), choline esters (acetylcholine, methacholine and carbachol), cholinomimetics (muscarine and arecoline) and polypeptides which stimulate peristalsis (eledoisin and angiotensin) antagonize the peristaltic block caused by leu-enkephalin. On the other hand, nicotinic ganglionic stimulants (nicotine and dimethylphenylpiperazine) as well as muscarinic ganglionic stimulants (McN-A-343 and AHR-602) do not restore the peristaltic reflex abolished by leu-enkephalin. Thus the inhibitory effect of leu-enkephalin is due mainly to an action on myenteric ganglia as well as on axon terminals of the myenteric plexus subserving the peristaltic reflex. The inhibitory action of leu-enkephalin may be ascribed to the opiate as well as to the cholinoceptive sites in the nervous elements in the myenteric plexus. The blocking action of leu-enkephalin is not associated with ganglionic muscarinic M-1 receptors as well as with ganglionic nicotinic receptors in the myenteric plexus of the guinea-pig isolated ileum.     

Muscarinic receptors couple to modulation of nicotinic ACh receptor desensitization in myenteric neurons

Signaling mechanisms coupled to activation of different neurotransmitter receptors interact in the enteric nervous system. ACh excites myenteric neurons by activating nicotinic ACh receptors (nAChRs) and muscarinic receptors expressed by the same neurons. These studies tested the hypothesis that muscarinic receptor activation alters the functional properties of nAChRs in guinea pig small intestinal myenteric neurons maintained in primary culture. Whole cell patch-clamp techniques were used to measure inward currents caused by ACh (1 mM) or nicotine (1 mM). Currents caused by ACh and nicotine were blocked by hexamethonium (100 microM) and showed complete cross desensitization. The rate and extent of nAChR desensitization was greater when recordings were obtained with ATP/GTP-containing compared with ATP/GTP-free pipette solutions. These data suggest that ATP/GTP-dependent mechanisms increase nAChR desensitization. The muscarinic receptor antagonist scopolamine (1 microM) decreased desensitization caused by ACh but not by nicotine, which does not activate muscarinic receptors. Phorbol 12,13-dibutyrate (10-100 nM), an activator of protein kinase C (PKC), but not 4-alpha-phorbol 12-myristate 13-acetate (a PKC inactive phorbol ester), increased nAChR desensitization caused by ACh and nicotine. Forskolin (1 microM), an activator of adenylate cyclase, increased nAChR desensitization, but this effect was mimicked by dideoxyforskolin, an adenylate cyclase inactive forskolin analog. These data indicate that simultaneous activation of nAChRs and muscarinic receptors increases nAChR desensitization. This effect may involve activation of a PKC-dependent pathway. These data also suggest that nAChRs and muscarinic receptors are coupled functionally through an intracellular signaling pathway in myenteric neurons.     

PKC-epsilon translocation in enteric neurons and interstitial cells of Cajal in response to muscarinic stimulation

Interstitial cells of Cajal in the deep muscular plexus (ICC-DMP) of the small intestine express excitatory neurotransmitter receptors. We tested whether ICC-DMP are functionally innervated by cholinergic neurons in the murine intestine. Muscles were stimulated by intrinsic nerves and ACh and processed for immunohistochemistry to determine these effects on PKC-epsilon activation. Under control conditions, PKC-epsilon-like immunoreactivy (PKC-epsilon-LI) was only observed in myenteric neurons within the tunica muscularis. Electrical field stimulation or ACh caused translocation of neural PKC-epsilon-LI from the cytosol to a peripheral compartment. After stimulation, PKC-epsilon-LI was found in spindle-shaped cells in the DMP. These cells were identified as ICC-DMP by Kit-LI and vimentin-LI. PKC-epsilon-LI in ICC-DMP and translocation of PKC epsilon-LI in neurons were blocked by tetrodotoxin or atropine, suggesting that these responses were due to activation of muscarinic receptors. Western blots also confirmed translocation of PKC-epsilon-LI. In conclusion, PKC-epsilon translocation is linked to muscarinic receptor activation in ICC-DMP and a subpopulation of myenteric neurons. These studies demonstrate that ICC-DMP are functionally innervated by excitatory motoneurons.     

5-Hydroxytryptamine2 and 5-hydroxytryptamine3 receptors mediate serotonin-induced short-circuit current in pig jejunum

The purpose of this study was to determine the effect of methysergide, ketanserin, granisetron, cisapride, and renzapride on serotonin 5-hydroxytryptamine-evoked short-circuit current in muscle and myenteric plexus-stripped pig jejunum using the Ussing chamber technique. Ketanserin, granisetron, cisapride, and renzapride all reduced the 5-hydroxytryptamine-induced increase in short-circuit current by about 50%. Combination of ketanserin and granisetron only reduced the 5-hydroxytryptamine-induced peak increase in short-circuit current by 25%. Cisapride caused a small concentration-dependent increase in short-circuit current. Atropine and hexamethonium both almost completely suppressed the cisapride-induced peak increase in short-circuit current. Ketanserin, granisetron, methysergide, and renzapride did not alter the basal short-circuit current. These results suggest that 5-hydroxytryptamine elicits an increase in short-circuit current by activating epithelial and submucosal 5-hydroxytryptamine₂ and 5-hydroxytryptamine₃ receptor subtypes. Furthermore, the short-circuit current-increasing effect of cisapride, is due to activation of at least muscarinic and nicotinic receptors.Abbreviations 5-HT 5-hydroxytryptamine, serotonin - AUC area under the curve - EC enterochromaffin - ENS enteric nervous system - GI gastrointestinal - MW molecular weight - 5-HTP-DP N-acetyl-5-hydroxytryptophyl-5-hydroxytrytophan amide - SSC short-circuit current - TTX tetrodotoxin     

Chronic decentralization of the heart differentially remodels canine intrinsic cardiac neuron muscarinic receptors

The objective of the study was to determine if chronic interruption of all extrinsic nerve inputs to the heart alters cholinergic-mediated responses within the intrinsic cardiac nervous system (ICN). Extracardiac nerve inputs to the ICN were surgically interrupted (ICN decentralized). Three weeks later, the intrinsic cardiac right atrial ganglionated plexus (RAGP) was removed and intrinsic cardiac neuronal responses were evaluated electrophysiologically. Cholinergic receptor abundance was evaluated using autoradiography. In sham controls and chronic decentralized ICN ganglia, neuronal postsynaptic responses were mediated by acetylcholine, acting at nicotinic and muscarinic receptors. Muscarine- but not nicotine-mediated synaptic responses that were enhanced after chronic ICN decentralization. After chronic decentralization, muscarine facilitation of orthodromic neuronal activation increased. Receptor autoradiography demonstrated that nicotinic and muscarinic receptor density associated with the RAGP was unaffected by decentralization and that muscarinic receptors were tenfold more abundant than nicotinic receptors in the right atrial ganglia in each group. After chronic decentralization of the ICN, intrinsic cardiac neurons remain viable and responsive to cholinergic synaptic inputs. Enhanced muscarinic responsiveness of intrinsic cardiac neurons occurs without changes in receptor abundance.     

Inhibitory effect of hemicholinium-3 on presynaptic nicotinic acetylcholine receptors located on the terminal region of myenteric motoneurons

Previously we have demonstrated the presence of presynaptic nicotinic acetylcholine receptors on the terminals of myenteric neurons in Auerbach's plexus of guinea-pig ileum. During these studies we observed, that the presence of hemicholinium-3, an inhibitor of the high affinity choline uptake significantly influences the contraction of the longitudinal muscle strip preparation. Our aim was to investigate the neurochemical background of this effect and quantitatively characterize the action of HC-3. We studied the effect of HC-3 on epibatidine- and electrical stimulation-evoked contraction and release of [3H]acetylcholine from the guinea-pig longitudinal muscle strip preparation. We found that in the presence of tetrodotoxin, when the contribution of somatodendritic nicotinic acetylcholine receptors to the response was prevented due to the inhibition of axonal conduction, HC-3 inhibited the epibatidine-evoked contraction and [3H]acetylcholine release in the submicromolar range (IC50 = 897 nM and IC50 = 693 nM, respectively), whereas the electrical stimulation-evoked contraction was not affected by HC-3, and the release of [3H]acetylcholine was apparently enhanced. Our data indicate that HC-3 inhibits the presynaptic nicotinic acetylcholine receptors of myenteric neurons. Since these receptors play an important role in the regulation of cholinergic neurotransmission in the enteric nervous system, the use of HC-3 in [3H]acetylcholine release experiments might bias the interpretation of data.     

Involvement of nitric oxide with activation of Toll-like receptor 4 signaling in mice with dextran sodium sulfate-induced colitis

Ulcerative colitis is an inflammatory bowel disease characterized by acute inflammation, ulceration, and bleeding of the colonic mucosa. Its cause remains unknown. Increases in adhesion molecules in vascular endothelium, and activated neutrophils releasing injurious molecules such as reactive oxygen species, are reportedly associated with the pathogenesis of dextran sodium sulfate (DSS)-induced colitis. Nitric oxide (NO) production derived from inducible NO synthase (iNOS) via activation of nuclear factor κB (NF-κB) has been reported. It is also reported that stimulation of Toll-like receptor 4 (TLR4) by lipopolysaccharide can activate NF-κB. In this study, we investigated the involvement of NO production in activation of the TLR4/NF-κB signaling pathway in mice with DSS-induced colitis. The addition of 5% DSS to the drinking water of male ICR mice resulted in increases in TLR4 protein in colon tissue and NF-κB p65 subunit in the nuclear fraction on day 3, increases in colonic tumor necrosis factor-α on day 4, and increases in P-selectin, intercellular adhesion molecule-1, NO₂⁻/NO₃⁻, and nitrotyrosine in colonic mucosa on day 5. These activated inflammatory mediators and pathology of colitis were completely suppressed by treatment with a NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, as well as an iNOS inhibitor, aminoguanidine. Conversely, a NO-releasing compound, NOC-18, increased TLR4 levels and nuclear translocation of NF-κB p65 and exacerbated mucosal damage induced by DSS challenge. These data suggest that increases in TLR4 expression induced by drinking DSS-treated water might be directly or indirectly associated with NO overproduction.     

大鼠回肠肌间神经丛一氧化氮合酶和乙酰胆碱酯酶双重显示法研究

本文用一氧化氮合酶和乙酰胆碱酯酶双重显示法,对大鼠回肠肌间神经丛进行了组织化学观察,结果发现三种不同染色的神经元：（１）乙酰胆碱酯酶阳性神经元（占８２％）;（２）一氧化氮合酶阳性神经元（占１６％）;（３）一氧化氮合酶和乙酰胆碱酯酶阳性神经元（占２％）。以上结果提示,一氧化氮可以与乙酰胆碱共存于大鼠回肠肌间神经丛的少数神经元内。本文还对肠肌间神经丛内神经元的类型和一氧化氮的作用进行了讨论。     

Role of the "little brain" in the gut in water and electrolyte homeostasis

The enteric nervous system plays a key role in maintenance of body fluid homeostasis by regulating the transport of ions by the intestinal epithelium. The epithelial cells normally absorb large volumes of fluid and ions daily, but tonically active submucosal neurons continuously suppress ion transport and limit the absorptive capacity of the intestine. Specialized nerve endings detect chemical, osmotic, or thermal alterations of the luminal contents or mechanical activity of the gut wall and encode this information as action potentials that propagate along nerve processes to the ganglia. Information transfer within the ganglia occurs at nicotinic cholinergic or other synapses. Ion transport is altered when neurotransmitters released from motor neurons interact with receptors on epithelial cells to initiate stimulus-response coupling. The signals that transduce changes in epithelial ion transport are largely unknown, except for acetylcholine, but may include vasoactive intestinal peptide or other peptides. These trigger changes in intracellular messengers that influence the state of ionic channels in the epithelial cells and thereby inhibit absorptive processes or stimulate secretory mechanisms. When conservation of salt and water is necessary, command signals from the central nervous system, and perhaps from the myenteric ganglia, will shut down the synaptic circuits in the submucosal ganglia and enhance the absorptive capacity of the bowel.     

Reduced sensitivity of inducible nitric oxide synthase-deficient mice to chronic colitis.

BACKGROUND: Overproduction of nitric oxide by the inducible form of nitric oxide synthase (iNOS) has been implicated in colitis. Different authors have postulated both toxic and protective effects of nitric oxide (NO) in the pathophysiology of active inflammation. The objective of this study was to examine the role of iNOS in experimental chronic colitis using iNOS-deficient mice. METHODS: For induction of colitis, mice received three cycles of 2% of dextran sodium sulfate (DSS) (M.W. 40,000) treatment in drinking water. The degree of colonic inflammation, leukocyte infiltration, and the expression of cell adhesion molecules were determined. INOS expression and nitrotyrosine were also determined by immunohistochemistry. RESULTS: After DSS treatment, a moderate colitis with marked cell infiltration was observed. Intense expression of iNOS was observed on infiltrating cells as well as on the colonic mucosal epithelium in these animals. In the iNOS-deficient mice, tissue damage was significantly diminished. No iNOS or nitrotyrosine staining was found in iNOS-deficient mice. The number of infiltrating cells and the expression of mucosal adressin cell adhesion molecule-1 were significantly attenuated in the DSS-treated colon of iNOS-deficient mice. CONCLUSION: Induction of iNOS seems to act as a critical toxic effector molecule in the pathogenesis of chronic colonic inflammation.     

Differential effects of maturation on nicotinic- and muscarinic receptor-induced ion secretion in guinea pig distal colon

The incidence of constipation increases with age. This has been linked to age-related changes in the structure and function of myenteric neurons regulating intestinal motility; however, the role of submucous neurons is unknown. The aim of this study was to determine the effect of maturation on cholinergic receptor-induced ion secretion in guinea pig colon. Changes in the short-circuit current (Isc) and tissue conductance were monitored in muscle-stripped colonic segments from young (3-4-month-old) and mature (12-15-month-old) male guinea pigs. Thirty-one percent of colonic segments from young guinea pigs exhibited ongoing neural activity, which was absent in mature animals. Baseline Isc was significantly higher only in young guinea pig tissues with ongoing activity. Tissue conductance was similar in all tissues. Electrical field stimulation caused a biphasic increase in the Isc. At 15 V/10 Hz, only Peak 1 was attenuated, whereas both peaks were reduced in mature guinea pigs at 10 V/5Hz. 1,1, dimethyl-4-phenyl-piperazinium(DMPP)-induced ion secretion was blunted in mature guinea pigs. Atropine reduced the 1,1, dimethyl-4-phenyl-piperazinium response only in young guinea pigs. Carbachol-induced ion secretion was similar in tissues from both age groups. In conclusion, nicotinic receptor-induced secretion mediated by both cholinergic and noncholinergic secretomotor neurons was blunted; however, epithelial muscarinic receptor activity was unaltered during maturation.     

eNOS involved in colitis-induced mucosal blood flow increase

The role of NO in inflammatory bowel disease is controversial. Studies indicate that endothelial nitric oxide synthase (eNOS) might be involved in protecting the mucosa against colonic inflammation. The aim of this study was to investigate the involvement of nitric oxide (NO) in regulating colonic mucosal blood flow in two different colitis models in rats. In anesthetized control and colitic rats, the distal colon was exteriorized and the mucosa visualized. Blood flow (laser-Doppler flowmetry) and arterial blood pressure were continuously monitored throughout the experiments, and vascular resistance was calculated. Trinitrobenzene sulfonic acid (TNBS) or dextran sulfate sodium (DSS) was used to induce colitis. All groups were given the NOS inhibitor N(omega)-nitro-l-arginine (l-NNA) or the inducible NOS (iNOS) inhibitor l-N(6)-(1-iminoethyl)-lysine (l-NIL). iNOS, eNOS, and neuronal NOS (nNOS) mRNA in colonic samples were investigated with real-time RT-PCR. Before NOS inhibition, colonic mucosal blood flow, expressed as perfusion units, was higher in both colitis models compared with the controls. The blood flow was reduced in the TNBS- and DSS-treated rats during l-NNA administration but was not altered in the control group. Vascular resistance increased more in the TNBS- and DSS-treated rats than in the control rats, indicating a higher level of vasodilating NO in the colitis models. l-NIL did not alter blood pressure or blood flow in any of the groups. iNOS and eNOS mRNA increased in both colitis models, whereas nNOS remained at the control level. TNBS- and DSS-induced colitis results in increased colonic mucosal blood flow, most probably due to increased eNOS activity.