Biomedical subjects

R A Lefebvre

Publications and source records attributed to R A Lefebvre.

At least 19 recordsLinked to original sources

PubMed2005

Involvement of NO in gastric emptying of semi-solid meal in conscious pigs.

The influence of non-selective nitric oxide synthase (NOS) inhibition on gastric emptying of a semi-solid meal was studied in conscious pigs. Antroduodenal motility and fundic compliance were also assessed to evaluate the mechanisms at the origin of potential alteration in gastric emptying pattern. N(G)-nitro-L-arginine methyl ester (L-NAME; 20 mg kg(-1) i.v.) delayed gastric emptying (half-emptying time of 128.98 +/- 16.86 min vs 73.74 +/- 7.73 min after saline, P < 0.05, n = 6) as a result of decreased proximal gastric emptying. No changes were observed for distal gastric emptying as a result of unchanged antral motility. Similarly, no changes were noted on duodenal motor patterns either in the fasted or in the fed state. L-NAME decreased fundic compliance in fasted state (49 +/- 11 mL mmHg(-1) vs 118 +/- 15 mL mmHg(-1) after saline, P < 0.05, n = 6). As this phenomenon is expected to increase emptying rate, the gastroparesis induced by NOS inhibition is thus likely to originate from distal resistive forces. It is concluded that NO positively modulates gastric emptying.

Animals↗

PubMed2005

5-HT4 receptors located on cholinergic nerves in human colon circular muscle.

5-Hydroxytryptamine 4 (5-HT4) receptor agonists promote colonic propulsion. The alteration of circular muscle (CM) motility underlying this involves inhibition of contractility via smooth muscle 5-HT4 receptors and proximal colonic motility stimulation, the mechanism of the latter not having been characterized. Our aim was to identify and characterize a 5-HT4 receptor-mediated stimulation of human colon CM contractile activity. 5-HT4 receptor ligands were tested on electrical field stimulation (EFS)-induced contractions of human colonic muscle strips cut in the circular direction (called 'whole tissue' strips). Additionally, after incubation of tissues with [3H]-choline these compounds were tested on EFS-induced release of tritium in whole tissue strips and in 'isolated' CM strips, obtained by superficial cutting in the CM layer. Tetrodotoxin and atropine blocked EFS-induced contractions of whole tissue CM strips. Prucalopride (0.3 micromol L-1) evoked a heterogenous response on EFS-induced contraction, ranging from inhibition (most frequently observed) to enhancement. In the release experiments, EFS-induced tritium efflux was blocked by tetrodotoxin. Prucalopride increased EFS-induced tritium and [3H]-acetylcholine efflux in whole tissue and in isolated CM strips. All effects of prucalopride were antagonized by the selective 5-HT4 receptor antagonist GR113808. The results obtained indicate the presence of excitatory 5-HT4 receptors on cholinergic nerves within the CM of human colon.

Acetylcholine↗

PubMed2005

Use of plasma ionized calcium levels and Ca2+ substitution response patterns as prognostic parameters for ileus and survival in colic horses.

OBJECTIVE: Hypocalcaemia is a common finding in horses with enterocolitis and severe gastrointestinal disorders. The aims of this study were to investigate in colic horses 1)the parameters related to hypocalcaemia, 2)the influence of hypocalcaemia on outcome and 3)the possible beneficial effect of Ca2+ substitution. DESIGN: Randomized controlled trial. SETTING: Intensive care unit. PATIENTS: One hundred forty-four horses that were admitted with an acute abdomen during a 1.5 year period were enrolled and daily evaluated for clinical criteria and whole blood ionized Ca2+ levels. Colic horses with hypocalcaemia were randomly assigned to receive Ca2+. INTERVENTIONS: Analysis of heparinised whole blood samples. Horses that were assigned to be treated received 400 mEq Ca2+ diluted in 10L of Ringer's lactate solution every 24 h until low reference range limits were obtained or until death. MEASUREMENTS AND MAIN RESULTS: 88% of all colic patients showed blood ionized Ca2+ levels below the reference range at the time of admission. Multivariable analysis revealed that the presence of reflux, signs of endotoxaemia, increased Packed Cell Volume (PCV), alkalinization of pH and the interaction PCV/pH all predispose colic horses to low ionized Ca2+ levels at the time of admission. The Odds for developing ileus during hospitalization are +/- 11.94 times larger for horses in the "very low" calcaemia interval, in comparison with normocalcaemic horses. The Odds for fatal outcome are respectively +/- 9.82 and 8.33 times larger for horses in the "very low" and "low" calcaemia interval. Ca2+ substitution increased the probability of survival, provided that Ca2+ levels could be normalized. The lack of an upward calcaemia response, despite repetitive Ca2+ substitutions, can be guarded as a poor ominous sign. CONCLUSIONS: Hypocalcaemia in colic horses is of prognostic relevance both with regard to survival as to the probability of development of ileus during hospitalization. This study shows the importance of routine measurement of ionized calcium levels in colic horses. Moreover, correction of hypocalcaemia seems to improve clinical outcome.

Abdomen, Acute↗

PubMed2004

Nitrergic-purinergic interactions in rat distal colon motility.

Responses of rat distal colon circular muscle strips to exogenous nitric oxide (NO) and adenosine 5'-triphosphate (ATP) and to electrical field stimulation (EFS) were assessed in the absence/presence of various agents that interfere with nitrergic-purinergic pathways. Exogenous NO (10-6 to 10-4 mol L-1) elicited concentration-dependent, tetrodotoxin (TTX)-insensitive relaxations. The soluble guanylyl-cyclase (sGC) inhibitor 1H[1,2,4,]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reduced duration and amplitude; the small conductance Ca2+-sensitive K+ (SK)-channel blocker apamin (APA) only shortened the relaxations. ODQ + APA showed a marked inhibitory effect on duration and amplitude. TTX, APA, the NO-synthase inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME) and the purinergic receptor P2Y antagonist Reactive Blue 2 (RB2) shortened the relaxations by exogenous ATP (10-3 mol L-1) but did not influence the amplitude. ODQ had no effect. TTX + l-NAME did not yield a more pronounced inhibitory effect than TTX alone. The effect of ATP-gamma-S was similar to that of ATP. Electrical field stimulation (EFS) (40 V, 0.05 ms, 0.5-4 Hz for 30 s) yielded TTX-sensitive relaxations that were not altered by l-NAME, ODQ or RB2. APA shortened the relaxations. l-NAME + APA nearly abolished these relaxations. ODQ + APA and RB2 +l-NAME reduced the duration. These results suggest that distinct sets of small conductance SK-channels are involved in the amplitude and the duration of the relaxations and that NO increases their sensitivity to NO and ATP via guanosine 3',5'-cyclic monophosphate (cGMP). ATP elicits relaxations via P2Y receptors with subsequent activation of SK-channels and induces neuronal release of NO. Both nitrergic and purinergic pathways must be blocked to inhibit EFS-induced relaxations.

Adenosine Triphosphate↗

PubMed2004

Interaction of NO and VIP in gastrointestinal smooth muscle relaxation.

Gastrointestinal (GI) smooth muscle cell activity is controlled by contractile cholinergic neurons and relaxant non-adrenergic non-cholinergic (NANC) neurons in the myenteric plexus between the circular and longitudinal muscle layer. Decreased or increased NANC relaxation might be involved in the pathophysiology of functional GI motility disorders. Vasoactive intestinal polypeptide (VIP) and nitric oxide (NO) are the primary inhibitory NANC neurotransmitters. As classic neurotransmitters, VIP is stored in vesicles in the nerve endings, while NO is synthetized on demand by the neuronal isoform of NO synthase (nNOS). The VIP/nNOS co-localization in myenteric neurons, reported for various regions of the GI tract in different species, suggests that VIP and NO are co-transmitters. At the presynaptic level, VIP and NO can induce each others release. Most clear-cut evidence for this mechanism was obtained in isolated myenteric ganglia where VIP induced NO release, and NO facilitated VIP release. At the postsynaptic level, many studies support that VIP and NO are parallel co-transmitters, acting via the adenylate cyclase/3'5' adenosine cyclic monophosphate (cAMP) and guanylate cyclase/3'5' cyclic guanosine monophosphate pathway respectively. Mainly based on results obtained in isolated GI smooth muscle cells, a serial postsynaptic VIP/NO interaction model was proposed, whereby VIP is the principle neurotransmitter, acting partially via a VPAC receptor and the adenylate cyclase/cAMP pathway but also by induction of muscular NO production. Recent results suggest that the capacity of VIP to release NO from isolated smooth muscle cells is related to the induction of inducible NOS (iNOS) in the cells during the isolation procedure. The relative contribution of NO and VIP to GI NANC relaxation differs upon tissue and nerve firing frequency, so that interference with either of them will lead to varying effects.

Animals↗

PubMed2003

In vivo characterization of 5-HT1A receptor-mediated gastric relaxation in conscious dogs.

Accumulating data have been published emphasizing the important role of 5-hydroxytryptamine (5-HT) receptors in proximal stomach relaxation. However, a proper in vivo characterization of 5-HT receptors mediating gastric relaxation is still missing. In the current study, we focus on the in vivo characterization of 5-HT1A receptors mediating relaxation of the proximal stomach in conscious dogs. Beagle dogs were equipped with a gastric fistula. In the conscious state, volume changes within an intragastric bag were measured at constant pressure by means of a barostat. Results are presented as the maximum volume increase after treatment (mean+/-s.e.m.). All drugs were injected intravenously. The 5-HT1A receptor agonist flesinoxan (10, 50, 100 and 150 microg kg-1) induced a dose-dependent relaxation of the canine proximal stomach (50+/-10, 230+/-51, 290+/-38 and 275+/-33 ml, respectively; n=9-11). The selective 5-HT1A receptor antagonist WAY-100635 dose-dependently inhibited the flesinoxan-induced relaxation. NG-nitro-l-arginine methyl ester did not affect this relaxation, suggesting that nitrergic nerves are not involved. After supradiaphragmatic vagotomy, the baseline of the intragastric volume was larger compared to that before vagotomy (317+/-50 vs 142+/-28 ml, respectively; n=5). Compensation for this by either reduction of the intraballoon pressure or infusion of a contractile dose of bethanechol did not establish a condition in which flesinoxan was able to relax the stomach. In contrast, nitroprusside induced a significant gastric relaxation when tone was increased by bethanechol. It is concluded that flesinoxan induces proximal gastric relaxation in conscious dogs via 5-HT1A receptors. The response is mediated through a vagal pathway without involvement of nitrergic nerves.

Animals↗

PubMed2003

Modulatory role of tachykinin NK1 receptor in cholinergic contraction of mouse trachea.

The role of the NK1 receptor in airway contraction induced by electrical field stimulation (EFS) was evaluated by comparing the response in NK1 receptor knockout mice (NK1R-/-) with that of NK1 receptor wild-type controls (WT). A frequency/response curve on tracheas from NK1R-/- mice and NK1R WT littermates was constructed. After incubation with [3H]choline, [3H]acetylcholine release upon EFS was measured by high-performance liquid chromatography and liquid scintillation counting. The effects of atropine (1 x 10(-6) M), tetrodotoxin (1 x 10(-6) M) and a specific NK1R antagonist (SR140333, 1 x 10(-8) M) were studied, as well as the effects of substance P (1 x 10(-5) M) on precontracted tracheas. Upon EFS, NK1R-/- mice had a significant lower trachea contractility than the NK1R WT animals, accompanied with less [3H]acetylcholine release. Pretreatment with atropine or tetrodotoxin abolished the EFS-induced contraction in both strains. Pretreatment with the NK1R antagonist SR140333 significantly reduced the contractility in the NK1R WT but not in the NK1R-/- mice. Substance P caused a small contraction in both NK1R WT and NK1R-/- mice. Substance P induced a relaxation in precontracted tracheas in NK1R WT but not in NK1R-/- mice. The data presented here provide direct evidence that the NK1 receptor augments cholinergic neurotransmission in mouse trachea.

Animals↗

PubMed2002

Influence of antioxidant depletion on nitrergic relaxation in the pig gastric fundus.

1. The hypothesis that endogenous tissue antioxidants might explain the inability of the superoxide generators 6-anilino-5,8-quinolinedione (LY83583) and hydroquinone (HQ) and of the NO-scavengers hydroxocobalamin (HC) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) to affect nitrergic neurotransmission in the porcine gastric fundus was tested by selective pharmacological depletion of respectively Cu/Zn superoxide dismutase (Cu/Zn SOD) and reduced glutathione (GSH) in circular smooth muscle preparations. 2. Diethyldithiocarbamate (DETCA; 3x10(-3) M), which almost completely abolished tissue Cu/Zn SOD activity, had no effect per se on nitrergic relaxations induced by either electrical field stimulation (EFS; 4 Hz, 10 s) or exogenous nitric oxide (NO; 10(-5) M). In these DETCA-treated tissues however, electrically-induced nitrergic relaxations became sensitive to inhibition by LY83583 (10(-5) M) or HC (10(-4) M), but not by HQ (10(-4) M) or c-PTIO (10(-4) M); only for the combination of DETCA plus LY83583, this inhibition was partially reversed by exogenous Cu/Zn SOD (1000 u ml(-1)). 3. Immunohistochemical analysis of porcine gastric fundus revealed a 100% colocalization of Cu/Zn SOD and neuronal nitric oxide synthase (nNOS) in the intrinsic neurons of the myenteric plexus. 4. Buthionine sulphoximine (BSO; 10(-3) M) in the absence or presence of LY83583 (10(-5) M) or HC (10(-4) M) did not alter nitrergic relaxations, although it reduced per se the tissue GSH content to 62% of control. 5. Pharmacological depletion studies, corroborated by immunohistochemical data, thus suggest a role for Cu/Zn SOD but not for GSH in nitrergic neurotransmission in the porcine gastric fundus.

Aminoquinolines↗

PubMed2002

Pharmacological characterization of the 5-HT receptors mediating contraction and relaxation of canine isolated proximal stomach smooth muscle.

1. We aimed to characterize 5-HT receptors mediating contraction and relaxation to 5-HT in dog proximal stomach longitudinal muscle (LM) strips. 2. Of the tryptamine analogues tested, 5-HT was the most potent contractile agent at basal length, while 5-CT was the most potent relaxant of PGF(2alpha)-induced contraction. Neither the contractions to 5-HT, nor the relaxations to 5-CT were influenced by tetrodotoxin, illustrating that action potential propagation is not involved. 3. The 5-HT-induced contraction was antagonized by mesulergine (0.03 to 0.3 microM) and ketanserin (2 - 20 nM), but the antagonism was not of a simple competitive nature, indicating multiple receptor involvement. Ketanserin (3 to 30 nM) and mesulergine (30 nM) competitively antagonized the alpha-Me-5-HT-induced contraction (pK(B): 8.83+/-0.09 and pA(2): 8.25+/-0.06 respectively). These affinity values are in line with literature affinities of ketanserin and mesulergine at 5-HT(2A) receptors in various bioassays. 4. The 5-CT-induced inhibition of PGF(2alpha)-induced contraction was competitively antagonized by mesulergine (pK(B) estimate: 8.52+/-0.12) and by the selective 5-HT(7) receptor antagonist SB-269970 (pK(B) estimate: 9.36+/-0.14). Both pK(B) estimates are in line with literature affinities of these compounds for 5-HT(7) receptors. Mesulergine (30 nM) and SB-269970 (10 nM) shifted the relaxant curve to 5-HT parallel to the right in the presence of ketanserin (0.3 microM) (pA(2) estimates of 8.08+/-0.10 and 8.75+/-0.14 respectively), indicative of 5-HT(7) receptor involvement. 5. It is concluded that 5-HT induces dog proximal stomach (LM) contraction via smooth muscle 5-HT(2A) receptors and relaxation via smooth muscle 5-HT(7) receptors.

Animals↗

PubMed2002

Smooth muscle 5-HT2A receptors mediating contraction of porcine isolated proximal stomach strips.

1. The aim of this study was to characterize the 5-HT receptors involved in the 5-HT-induced contraction of longitudinal muscle (LM) strips of porcine proximal stomach. This was done in a classical organ bath set-up for isotonic measurement. 2. The concentration-contraction curve to 5-HT was not modified by 5-HT(3) and 5-HT(4) receptor antagonism. Methysergide, ketanserin and mesulergine antagonized the curve to 5-HT. Concomitantly, increasing concentrations of ketanserin and mesulergine progressively revealed a biphasic nature of the 5-HT curve. Ketanserin antagonized the low-affinity receptor while it did not modify the high-affinity receptor. 3. Tetrodotoxin did not influence the concentration-contraction curve to 5-HT neither in the absence nor presence of ketanserin, indicating that nerves are not involved. 4. Ketanserin competitively antagonized the monophasic concentration-response curve to alpha-Methyl-5-HT, yielding a Schild slope that was not significantly different from unity. After constraining the Schild slope to unity, a pK(B) estimate of 8.23+/-0.90 was obtained. This affinity estimate of ketanserin closely approximates previously reported affinities at 5-HT(2A) receptors. 5. In the presence of ketanserin (0.1 microM; exposing the high-affinity receptor), a wide range of 5-HT receptor antagonists covering all 5-HT receptors known, was tested. Only methysergide and ritanserin inhibited the response to 5-HT, thus expressing affinity for the high-affinity receptor. This did not reveal the identity of the receptor involved. 6 It can be concluded that 5-HT induces pig proximal stomach (LM) contraction via 5-HT(2A) receptors located on smooth muscle. A ketanserin-insensitive phase of contractions could not be characterized between the actually known classes of 5-HT receptors with the pharmacological tools that were used.

Animals↗

PubMed2002

Pharmacological characterization of the nitrergic innervation of the stomach.

Proximal gastric relaxation is a vago-vagal reflex upon food intake. The efferent neurons involved at the level of the stomach are nonadrenergic noncholinergic. Deficient proximal gastric relaxation is observed in a portion of patients with functional dyspepsia, while exaggerated relaxation might contribute to the development of gastroesophageal reflux disease via triggering of transient lower esophageal sphincter relaxations. Nitric oxide (NO) is mediating, together with vasoactive intestinal polypeptide (VIP) as parallel cotransmitter, the nonadrenergic noncholinergic neurotransmission of the proximal stomach. Evidence for a sequential link between VIP as neurotransmitter and muscular NO generation was obtained when studied in isolated gastric smooth muscle cells; inducible NO synthase seems expressed. The endogenous gastric nitrergic neurotransmitter is not sensitive to superoxide anion generators and NO scavengers, that reduce the relaxation to exogenous NO. This is not due to the release of a nerve-derived hyperpolarizing factor in addition of NO, nor to binding to thiols, but Cu/Zn superoxide dismutase is involved in the protection of endogenous NO versus superoxide anions and scavenging. The release of NO from gastric nitrergic neurons is not sensitive to negative feedback but is inhibited via presynaptic alpha 2-adrenoceptors. Nitric oxide functionally antagonizes acetylcholine in the smooth muscle cells but does not influence the release of acetylcholine at the cholinergic varicosities. Stimulating or inhibiting the gastric nitrergic neurons might be a target for drug therapy in functional dyspepsia or gastro-esophageal reflux, respectively.

Dyspepsia↗

PubMed2001

In vitro and in vivo evaluation of starch-based hot stage extruded double matrix systems.

The objective of developing a double matrix system consisting of a hot stage extruded starch pipe surrounding a hot stage extruded and drug-containing starch core, was to obtain a monolithic matrix system applicable in the domain of sustained drug release. The behaviour of the systems was evaluated through dissolution testing and through a randomised crossover bioavailability study on nine male volunteers. All double matrix systems showed in vitro a nearly constant drug release profile after an initial slower release phase of 4 h. This initial slower release phase was avoided by loading the starch pipe with a small amount of drug. The in vitro dissolution profiles did not reflect the in vivo behaviour of the systems. Although the in vitro dissolution profiles of two selected double matrix systems were slower than the profile of a commercially available sustained release formulation, they performed less well in vivo. However, the t75%Cmax ratios of the evaluated double matrix systems versus a reference immediate release formulation indicated an acceptable sustained release behaviour, superior to that of hot stage extruded starch-based single matrices. Therefore the disclosed drug delivery systems could be applied in the domain of sustained drug delivery. Besides, the continuous production of the double matrix systems offers an advantage from a manufacturing point of view.

Adult↗

PubMed2001

Effect of TNBS-induced morphological changes on pharmacological contractility of the rat ileum.

Intraluminal instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS) in ethanol is a classical model of colitis in the rat. Little is known about the time-related effect of TNBS on the contractility and morphology of the rat ileum. After 36 h, TNBS induced acute ileitis. Spontaneous activity of longitudinal muscle strips was decreased, as were receptor- and nonreceptor-mediated contractions and contractions induced by electrical stimulation. After 1 week, mucosal integrity was restored, although the thickness of both mucosal and muscle layers was increased. Spontaneous activity, receptor- and nonreceptor-mediated contractions and electrically induced contractions of longitudinal muscle strips were increased due to hypertrophy and hyperplasia of smooth muscle cells. This was confirmed in the contractility study of individual muscle cells. Functional alterations after 1 week were restricted to a decreased response to substance P. TNBS-ileitis in the rat lacks a chronic phase and is accompanied by functional hypocontractility of longitudinal smooth muscle cells during the acute inflammation, whereas the contractility of the longitudinal muscle layer is increased in the postinflammation phase due to structural alterations. There is a selective inhibition of the response to substance P in the postinflammation phase.

Acetylcholine↗

PubMed2001

Influence of nitric oxide donors and of the alpha(2)-agonist UK-14,304 on acetylcholine release in the pig gastric fundus.

This study in circular muscle strips of the pig gastric fundus aimed to measure the release of acetylcholine directly and to investigate whether NO and alpha(2)-adrenoceptor agonists can modulate acetylcholine release from cholinergic neurones. After incubation of the tissues with [(3)H]-choline, basal and electrically induced release of tritium and [(3)H]-acetylcholine were analyzed in a medium containing physostigmine (10(-5) M) as well as atropine (10(-6) M). The NO synthase inhibitor L-N(G)-nitroarginine methyl ester (3x10(-4) M), and the NO donors sodium nitroprusside (10(-5) M) and 3-morpholino-sydnonimine (10(-5) M) did not influence the basal release nor the electrically evoked release, indicating that NO does not modify [(3)H]-acetylcholine release. The alpha(2)-adrenoceptor agonist UK-14,304 (10(-5) M) significantly inhibited the electrically evoked release of [(3)H]-acetylcholine, and this effect was prevented by the alpha(2)-adrenoceptor antagonist rauwolscine (2x10(-6) M), suggesting that presynaptic alpha(2)-adrenoceptors are present on cholinergic neurones of the pig gastric fundus.

Acetylcholine↗

PubMed2001

5-HT(4) receptors mediating enhancement of contractility in canine stomach; an in vitro and in vivo study.

We aimed to study 5-HT(4) receptors in canine stomach contractility both in vivo and in vitro. In anaesthetized Beagle dogs, the selective 5-HT(4) receptor agonist prucalopride (i.v.) induced dose-dependent tonic stomach contractions under isobaric conditions, an effect that was antagonized by the selective 5-HT(4) receptor antagonist GR 125487 (10 microg kg(-1), i.v.). Electrical field stimulation (EFS) of corpus longitudinal muscle strips resulted in atropine- and tetrodotoxin-sensitive contractions (L-NOARG (0.1 mM) present in all organ bath solutions). Prucalopride increased these contractions (maximal response after single-dose addition (0.3 microM): 165% of initial value, or after cumulative addition: 188%). In the presence of methysergide (3 microM), 5-HT also increased EFS-contractions (after single-dose addition (0.3 microM): increase to 192%, after cumulative addition: 148%). The selective 5-HT(4) receptor antagonists GR 113808 (0.1 microM) or GR 125487 (10 nM) antagonized the prucalopride (0.3 microM)-induced contraction increments. When EFS-induced contractions were blocked by atropine or tetrodotoxin, prucalopride was ineffective. In the presence of methysergide (3 microM), the contraction increases to 5-HT (0.3 microM) were prevented by GR 113808 (0.1 microM). The prucalopride curve (pEC(50) 7.9) was shifted in parallel to the right by GR 113808 3 nM (pA(2) 9.4). In the presence of methysergide (3 microM), the curve to 5-HT (pEC(50) 8.1) was competitively antagonized by GR 113808, yielding a Schild slope of 0.8+/-0.2 (pK(B) of 9.1 with unit Schild slope). In corpus circular muscle strips, the prucalopride (0.3 microM)-induced augmentation of EFS-contractions (258%) was also prevented by GR 113808 (0.1 microM) (124%). In conclusion, the effects of 5-HT(4) receptor agonists on proximal stomach motor activity in vivo can be explained by an effect on 5-HT(4) receptors on cholinergic nerves within the gastric muscle wall.

Animals↗

PubMed2001

Antisense knockdown of inducible nitric oxide synthase inhibits the relaxant effect of VIP in isolated smooth muscle cells of the mouse gastric fundus.

1. Our previous results showed that the non-selective nitric oxide synthase (NOS) inhibitor L-N(G)-nitroarginine (L-NOARG) and the selective inducible NOS (iNOS) inhibitor N-(3-(acetaminomethyl)-benzyl)acetamidine (1400W) inhibited the relaxant effect of vasoactive intestinal polypeptide (VIP) in isolated smooth muscle cells of the mouse gastric fundus, suggesting the involvement of iNOS. The identity of the NOS isoform involved in the VIP-induced relaxation in isolated smooth muscle cells of the mouse gastric fundus was now further investigated by use of antisense oligodeoxynucleotides (aODNs) to iNOS. 2. Incubation of isolated smooth muscle cells with fluorescein isothiocyanate (FITC)-labelled aODNs showed that nuclear accumulation occurs quickly and reaches saturation after 60 min. The in vivo intravenous administration of aODNs to iNOS, 24 and 12 h before murine tumour necrosis factor alpha (mTNFalpha) challenge, significantly reduced the nitrite levels induced by the mTNFalpha challenge. 3. Intravenous administration of aODNs to iNOS in mice, 24 and 12 h before isolation of the gastric smooth muscle cells, decreased the inhibitory effect of the NOS inhibitors L-NOARG and 1400W on the relaxant effect of VIP, whereas neither saline nor sODNs had any influence. 4. Preincubation of the isolated smooth muscle cells with aODNs almost abolished the inhibitory effect of L-NOARG and 1400W on the VIP-induced relaxation, whereas sODNs failed. 5. These results illustrate that the inhibitory effect of NOS inhibitors in isolated smooth muscle cells of the mouse gastric fundus is due to inactivation of iNOS. iNOS, probably induced by the isolation procedure of the smooth muscle cells, seems involved in the relaxant effect of VIP in isolated gastric smooth muscle cells.

Amidines↗

PubMed2001

Characterization of the receptors involved in the 5-HT-induced excitation of canine antral longitudinal muscle.

1. We aimed to characterize the 5-HT receptors involved in the 5-HT-induced effect on electrically induced contractions of dog antrum longitudinal muscle in vitro. 2. In the presence of L-NOARG (0.1 mM), electrical field stimulation (EFS) induced atropine- and tetrodotoxin-sensitive contractions. Tetrodotoxin or atropine left any agonist tested ineffective. These EFS-induced contractions were on average enhanced by 5-HT (0.3 microM), however, pronounced variation in the response to 5-HT was observed. There were non-significant trends of the selective 5-HT3 receptor antagonist granisetron (1 microM), and methysergide (1 microM; preventing interactions of 5-HT with 5-HT1, 5-HT2, 5-ht5, 5-HT6 and 5-HT7 receptors) to increase the response to 5-HT. The selective 5-HT4 receptor antagonist GR 113808 (0.1 microM) displayed a non-significant trend to inhibit the 5-HT-induced increase. 3. Combination experiments with methysergide (1 microM), granisetron (1 microM) and GR 113808 (0.1 microM) revealed that the 5-HT (0.3 microM)-induced response consisted of (1) an excitatory component blocked by GR 113808, (2) excitatory and inhibitory components both blocked by methysergide. 4. The selective 5-HT4 receptor agonist prucalopride (0.3 microM) increased EFS-induced contractions, an effect prevented by GR 113808 (0.1 microM). 5. The increase of EFS-induced contractions by the preferential 5-HT2 receptor agonist alpha-Me-5-HT (0.3 microM) was antagonized by 5-HT2B receptor antagonists. 6. The 5-HT1/5-HT7 receptor agonist 5-carboxamidotryptamine (5-CT; 0.3 microM) inhibited EFS-induced contractions. This was prevented by methysergide (1 microM), the 5-HT7 receptor antagonist mesulergine (0.3 microM) and the selective 5-HT7 receptor antagonist SB-269970 (0.3 microM). 7. In the presence of GR 113808 (0.1 microM), alpha-Me-5-HT (1 microM) increased EFS-induced contractions. The 5-HT (0.3 microM)-induced inhibition of the stimulation by alpha-Me-5-HT was prevented by SB-269970 (0.3 microM). 8. In conclusion, dog antral longitudinal muscle is endowed with (1) excitatory neuronal 5-HT4 receptors and 5-HT2B receptors and (2) inhibitory smooth muscle 5-HT7 receptors.

Animals↗

PubMed2000

Subcellular localization of neuronal nitric oxide synthase in rat small intestine.

The subcellular localization of neuronal nitric oxide synthase (NOS I, EC 1.14.13.39) was investigated in the longitudinal muscle/myenteric plexus (LM/MP) preparation of rat small intestine. The presence of NOS I, inducible nitric oxide synthase (NOS II), and endothelial nitric oxide synthase (NOS III) was assessed after homogenization and low-speed centrifugation in a postnuclear supernatant by immunological detection after PAGE and Western blotting. Only NOS I was clearly present, whereas NOS II and NOS III were below detection limits. After high-speed centrifugation of the postnuclear supernatant, soluble and particulate fractions were obtained, and the presence of NOS I in these fractions was investigated by measurement of NOS I immunoreactivity and enzyme activity. We found that 90 +/- 1% of NOS I immunoreactivity and 97 +/- 1% of NOS enzyme activity were confined to the soluble fraction of the tissue. Further immunological analysis demonstrated that washing the particulate fraction revealed detectable amounts of NOS I only after concentration of the washing supernatant. Most particulate NOS I remained in the pellet and therefore represents cell organelle-associated enzyme. No NOS I immunoreactivity could be detected as a soluble protein within organelles of the cell. Particulate NOS I could in part be solubilized by Triton X-100 treatment, and the detection of Triton X-100-soluble NOS I was dependent on the antibody used. In conclusion, our results indicate that NOS I in the LM/MP preparation of rat small intestine is mainly soluble and that the particulate NOS I is partly an intrinsic membrane protein and can partly be solubilized by detergent treatment.

Animals↗