Biomedical subjects

R A Lefebvre

Publications and source records attributed to R A Lefebvre.

At least 37 records · Page 2Linked to original sources

PubMed2000

Interplay between nitric oxide and vasoactive intestinal polypeptide in the pig gastric fundus smooth muscle.

The aim of this study was to investigate the exact mechanism of interaction between nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) as inhibitory non-adrenergic non-cholinergic (NANC) neurotransmitters in isolated smooth muscle cells and smooth muscle strips of the pig gastric fundus. In isolated smooth muscle cells, the maximal relaxant effect of VIP (10(-9) M) was inhibited by 94% by the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine (L-NA, 10(-4) M) and by 85% by the inducible NOS (iNOS)-selective inhibitor N-(3-(aminomethyl)-benzyl)acetamide (1400W; 10(-6) M). The relaxant effect of VIP was reduced by more than 70% by the guanylyl cyclase inhibitor 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ; 10(-6) M), the glucocorticoid dexamethasone (10(-5) M) and three protein kinase A inhibitors: (R)-p-cyclic adenosine-3', 5'-monophosphothioate ((R)-p-cAMPS; 10(-6) M), ¿(8R,9S, 11S)-(-)-9-hydroxy-9-n-hexylester-8-methyl-2,3,9,10-tetrahydro-8, 11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo[a, g]cycloocta[cde]-trin-den-1-one¿ (KT5720; 10(-6) M) and N-(2-(p-bromo-cinnamylamino)ethyl))-5-isoquinoline sulfonamide dihydrochloride (H-89; 10(-5) M). In contrast, no influence of the NOS inhibitors, ODQ, dexamethasone, nor the protein kinase A inhibitors could be observed on the relaxant effect of VIP in smooth muscle strips. These data demonstrate that the experimental method completely changes the influence of NOS inhibitors on the relaxant effect of VIP in the pig gastric fundus. The isolation procedure of the smooth muscle cells might induce iNOS that can be activated by VIP.

Adenine↗

PubMed2000

Investigation of the interaction between nitric oxide and vasoactive intestinal polypeptide in the guinea-pig gastric fundus.

The interaction between nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) was investigated in isolated circular smooth muscle cells and strips of the guinea-pig gastric fundus. VIP induced a concentration-dependent inhibition of carbachol-induced contraction in smooth muscle cells with a maximum at 10(-6) M. The relaxation by 10(-6) M VIP was inhibited for 79.1+/-5.8% (mean+/-s.e. mean) by the NO-synthase (NOS) inhibitor L-N(G)-nitroarginine (L-NOARG; 10(-4) M) in a L-arginine reversible way. Also the inducible NOS (iNOS) selective inhibitor N-(3-(acetaminomethyl)-benzyl)acetamide (1400 W; 10(-6) M) inhibited the VIP-induced relaxation, but its inhibitory effect was not reversed by L-arginine. When cells were incubated with the guanylyl cyclase inhibitor 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ, 10(-6) M), the protein kinase A-inhibitor (R)-p-cyclic adenosine-3', 5'-monophosphothioate ((R)-p-cAMPS, 10(-6) M) and the glucocorticoid dexamethasone (10(-5) M), the relaxant effect of VIP was decreased by respectively 80.9+/-7.6, 77.0+/-11.6 and 87.1+/-4.5%. In circular smooth muscle strips of the guinea-pig gastric fundus, the VIP (10(-9) - 10(-7) M)-induced relaxations were not significantly influenced by 10(-4) M L-NOARG, 10(-6) M 1400 W, 10(-6) M ODQ and 10(-5) M dexamethasone. These results suggest that iNOS, possibly induced by the procedure to prepare the smooth muscle cells, is involved in the relaxant effect of VIP in isolated smooth muscle cells but not in smooth muscle strips of the guinea-pig gastric fundus. This study illustrates the importance of the experimental method when studying the influence of NOS inhibitors on the relaxation induced by VIP in gastrointestinal smooth muscle preparations.

Adenine↗

PubMed2000

Influence of bilirubin and other antioxidants on nitrergic relaxation in the pig gastric fundus.

1. The influence of several antioxidants (bilirubin, urate, ascorbate, alpha-tocopherol, glutathione (GSH), Cu/Zn superoxide dismutase (SOD) and the manganese SOD mimic EUK-8) on nitrergic relaxations induced by either exogenous nitric oxide (NO; 10(-5) M) or electrical field stimulation (4 Hz; 10 s and 3 min) was studied in the pig gastric fundus. 2. Ascorbate (5x10(-4) M), alpha-tocopherol (4x10(-4) M), SOD (300 - 1000 u ml(-1)) and EUK-8 (3x10(-4) M) did not influence the relaxations to exogenous NO. In the presence of GSH (5x10(-4) M), the short-lasting relaxation to NO became biphasic, potentiated and prolonged. Urate (4x10(-4) M) and bilirubin (2x10(-4) M) also potentiated the relaxant effect of NO. None of the antioxidants influenced the electrically evoked relaxations. 3. 6-Anilino-5,8-quinolinedione (LY83583; 10(-5) M) had no influence on nitrergic nerve stimulation but nearly abolished the relaxant response to exogenous NO. Urate and GSH completely prevented this inhibitory effect, while it was partially reversed by SOD and bilirubin. Ascorbate, alpha-tocopherol and EUK-8 were without effect. 4. Hydroquinone (10(-4) M) did not affect the electrically induced nitrergic relaxations, but markedly reduced NO-induced relaxations. The inhibition of exogenous NO by hydroquinone was completely prevented by urate and GSH. SOD and ascorbate afforded partial protection, while bilirubin, EUK-8 and alpha-tocopherol were ineffective. 5. Hydroxocobalamin (10(-4) M) inhibited relaxations to NO by 50%, but not the electrically induced responses. Full protection versus this inhibitory effect was obtained with urate, GSH and alpha-tocopherol. 6. These results strengthen the hypothesis that several endogenous antioxidant defense mechanisms, enzymatic as well as non-enzymatic, might play a role in the nitrergic neurotransmission process.

Aminoquinolines↗

PubMed2000

An improved in vitro bioassay for the study of 5-HT(4) receptors in the human isolated large intestinal circular muscle.

Recently, it was demonstrated that 5-HT induces relaxation of human colon circular muscle through activation of 5-HT(4) receptors and 5-HT(7) receptors. The aim of the current study was to develop a new in vitro bioassay of human colon that would facilitate the pharmacological analysis of 5-HT responses mediated solely by 5-HT(4) receptors. Contracting circular muscle strips with KCl (80 mM) yielded a stable contractile tension and, in contrast to muscarinic cholinoceptor agonists and histamine, a profound reduction of spontaneous contractility. This allowed the establishment of reproducible, fully-defined, agonist concentration-response curves by cumulative dosing. Under these conditions, 5-HT induced a concentration-dependent relaxation (pEC(50) 7.31, Hill slope 0.91). Neither methysergide (10 microM) nor granisetron (1 microM) affected the 5-HT-induced relaxation, suggesting that 5-HT(1), 5-HT(2), 5-HT(3), 5-ht(5), 5-HT(6) or 5-HT(7) receptors are not involved. The lack of effect of tetrodotoxin (0.3 microM) indicated a direct effect of 5-HT on the smooth muscle. The selective 5-HT(4) receptor antagonists GR 113808, GR 125487 and RS 39604 competitively antagonized the 5-HT-induced relaxation (pK(B) 9.43, 10.12 and 8.53, respectively). SB 204070 (1 nM) produced a rightward shift (pA(2) 10.34) and depression of the 5-HT curve. These affinity estimates are similar to those previously reported for 5-HT(4) receptors. The selective 5-HT(4) receptor agonists, prucalopride and R076186, induced relaxations (pEC(50) 7.50 and 7.57, respectively), that were blocked by GR 113808 (3 nM), yielding pA(2) estimates of 9.31 and 9.21, respectively. To summarise, in KCl (80 mM)-contracted muscle strips, 5-HT induces relaxation through activation of a homogeneous smooth muscle 5-HT(4) receptor population. This new bioassay allows the focused, pharmacological characterization of human colonic 5-HT(4) receptors in vitro.

Biological Assay↗

PubMed2000

Interaction of hypoxanthine/xanthine oxidase with nitrergic relaxation in the porcine gastric fundus.

The influence of hypoxanthine (HX)/xanthine oxidase (XO) on short-term [electrical field stimulation (EFS; 4 Hz) for 10 s and 3 min; bolus of exogenous NO (10(-5) M)] and long-term [EFS (4 Hz) and continuous NO-infusion for 20 min] nitrergic relaxations was investigated in circular muscle strips of the pig gastric fundus. HX (3x10(-4) M) / XO (64 mu ml(-1)) did not affect EFS for 10 s and 3 min; the short-lasting relaxation in response to a bolus of exogenous NO (10(-5) M) was changed into a biphasic relaxation with a small and short first phase followed by a larger and prolonged second phase. Cu/Zn superoxide dismutase (Cu/Zn SOD; 1000 u ml(-1)) and uricase (100 mu ml(-1)) respectively enhanced the amplitude of the first phase and diminished the amplitude of the second phase. Ascorbate (5x10(-4) M) and bilirubin (2x10(-4) M) prevented the prolonged component. Exposure to HX/XO during long-term EFS elicited a complete, stable reversal of relaxation starting after a delay. During continuous NO-infusion, HX/XO induced an immediate, complete but transient reversal. The antioxidants bilirubin, ascorbate, alpha-tocopherol, urate, glutathione and Cu/Zn SOD, the hydrogen peroxide degrading enzyme catalase, the hydroxyl radical scavengers dimethylsulphoxide and mannitol, and the cofactor flavin adenine dinucleotide did not influence the reversal induced by HX/XO during either EFS or NO-infusion. The cell-permeable manganese SOD mimetic EUK-8 modified the stable reversal during long-term EFS into a transient one. The results suggest that a nitrated uric acid derivative is responsible for the prolonged second phase in the relaxation to a bolus of exogenous NO in the presence of HX/XO. The exact underlying mechanism of the reversal induced by HX/XO during sustained relaxation remains unclear.

Animals↗

PubMed2000

5-HT(4) receptors on cholinergic nerves involved in contractility of canine and human large intestine longitudinal muscle.

5-HT(4) receptors mediate circular muscle relaxation in both human and canine large intestine, but this phenomenon alone can not explain the improvement in colonic motility induced by selective 5-HT(4) receptor agonists in vivo. We set out to characterize 5-HT(4) receptor-mediated effects in longitudinal muscle strips of canine and human large intestine. Electrical field stimulation (EFS) was applied providing submaximal isotonic contractions. L-NOARG (0.1 mM) was continuously present in the organ bath to preclude nitric oxide-induced relaxation to EFS. The selective 5-HT(4) receptor agonist prucalopride (0.3 microM) enhanced EFS-evoked contractions, that were antagonized in both preparations by the selective 5-HT(4) receptor antagonist GR 113808 (0.1 microM). The prucalopride-induced increase was present in canine ascending and descending colon, but absent in rectum. Regional differences in response to prucalopride were not observed in human ascending and sigmoid colon and rectum. Incubation with atropine (1 microM) or tetrodotoxin (0.3 microM) inhibited EFS-induced contractions, which were then unaffected by prucalopride (0.3 microM) in both tissues. In the presence of methysergide (3 microM; both tissues) and granisetron (0.3 microM; only human tissues), 5-HT (0.3 microM) enhanced EFS-induced contractions, an effect that was antagonized by GR 113808 (0.1 microM). In the presence of atropine or tetrodotoxin, EFS-induced contractions were inhibited, leaving 5-HT (0.3 microM) ineffective in both preparations. This study demonstrates for the first time that in human and canine large intestine, 5-HT(4) receptors are located on cholinergic neurones, presumably mediating facilitating release of acetylcholine, resulting in enhanced longitudinal muscle contractility. This study and previous circular muscle strip studies suggest that 5-HT(4) receptor agonism facilitates colonic propulsion via a coordinated combination of inhibition of circumferential resistance and enhancement of longitudinal muscle contractility.

Animals↗

PubMed2000

Paroxetine affects metoprolol pharmacokinetics and pharmacodynamics in healthy volunteers.

OBJECTIVE: To investigate the effect of multiple-dose paroxetine intake on the stereoselective pharmacokinetics and the pharmacodynamics of metoprolol. METHODS: We conducted an open trial with two sessions in eight healthy male volunteers. Racemic metoprolol (100 mg single oral dose) was administered before and after paroxetine treatment (20 mg/day for 6 days). The (R)- and (S)-metoprolol pharmacokinetics, metoprolol metabolic ratio (MR), exercise heart rate and blood pressure were assessed for 12 (pharmacodynamic data) to 24 (pharmacokinetic data) hours after each metoprolol intake. RESULTS: Paroxetine treatment increased the mean area under the plasma concentration-time curve extrapolated to infinity (AUC) of (R)- and (S)-metoprolol significantly (169 to 1,340 ng x h/mL [P < .001] and 279 to 1,418 ng x h/mL [P < .001], respectively), with an approximately twofold increase in both maximum plasma concentration and terminal elimination half-life. Furthermore, the (S)/(R) AUC ratio was significantly decreased, from 1.72 to 1.07 (P < .001). The mean metoprolol MR was significantly increased, from 0.17 to 5.69 (P < .05). The AUC of the metoprolol-induced decrease in exercise heart rate versus time curve was increased, with 46% (P < .01) after multiple-dose paroxetine intake, reaching significance from 6 hours after metoprolol intake, illustrating a more sustained beta-blockade. Similar results were obtained for the effect on exercise systolic blood pressure. Multiple-dose metoprolol administration combined with paroxetine can lead to an accumulation of the beta-blocking (S)-enantiomer of metoprolol, possibly resulting in unacceptable bradycardia, loss of cardioselectivity, or both. CONCLUSION: Multiple-dose paroxetine intake affects both metoprolol pharmacokinetics and pharmacodynamics and suggests that when paroxetine is added to an ongoing metoprolol therapy, caution is warranted and a reduction of the metoprolol dose may be required to prevent undesired adverse effects.

Adrenergic beta-Antagonists↗

PubMed1999

Bioavailability of starch based hot stage extrusion formulations.

The aim of the study was to develop a starch based hot stage extrusion formulation for controlled drug delivery and to evaluate its in vivo behavior. The extrusion mixture consisted of 53% corn starch as the matrix forming agent, 15% sorbitol as a plasticizer, 30% theophylline monohydrate as the model drug and 2% glyceryl monostearate as a lubricant. The extrudates were produced by means of a corotating twin screw extruder of APV Baker equipped with a twin screw powder feeder and a 3-mm cylindrical die. During extrusion 20% water (based on the wet mass) was added to the powder mixture. The extrudates were dried in an oven at 60 degrees C during 48 h, cut and filled out in hard gelatine capsules, in a way that the content of two capsules corresponded with a dose of 300 mg anhydrous theophylline. The dissolution profile of the experimental dosage form was retarded with a drug release of around 80% in 8 h. The in vivo behavior of the experimental formulation was evaluated in a randomized crossover design study (n=8) with a commercially available multiple unit sustained release product as the reference formulation. The plasma samples were analyzed by a validated HPLC-UV method with solid phase extraction for the sample preparation. It was clear that the experimental formulation exhibited sustained release behavior, but that it performed less well than the multiple unit dosage form.

Adult↗

PubMed1999

Pharmacological characterization of 5-HT4 receptors mediating relaxation of canine isolated rectum circular smooth muscle.

This study aimed to characterize for the first time in vitro 5-HT4 receptors in the canine gastrointestinal tract. For this purpose, we used circular muscle strips of the canine isolated rectum. In the presence of methysergide (60 microM), 5-HT induced relaxation of methacholine (1 microM)-precontracted muscle strips, yielding a monophasic sigmoidal concentration-relaxation curve (pEC50 7.2+/-0.07). Tetrodotoxin (0.3 microM) did not affect the curve to 5-HT, suggesting the inhibitory 5-HT receptor is located on the smooth muscle. Granisetron (0.3 microM) did also not affect the curve to 5-HT, which excludes the 5-HT3 receptor mediating the relaxation to 5-HT. The presence of methysergide rules out the involvement of 5-HT1, 5-HT2 or 5-HT7 receptors. 5-HT, the selective 5-HT4 receptor agonists R076186, prucalopride (R093877) and SDZ HTF-919 and the 5-HT4 receptor agonists cisapride and 5-MeOT relaxed the muscle strips with a rank order of potency R076186 = 5-HT > cisapride > prucalopride > or = SDZ HTF-919 > 5-MeOT. The selective 5-HT4 receptor antagonists GR 125487, RS 39604 and GR 113808 competitively antagonized the relaxations to 5-HT, yielding pK(B) estimates of 9.7, 7.9 and 9.1, respectively. The selective 5-HT4 receptor antagonist SB 204070 shifted the curve to 5-HT rightward and depressed the maximal response (apparent pA2 10.6). GR 113808 (10 nM) produced a parallel rightward shift of the curve to the selective 5-HT4 receptor agonists R076186 (pA2 8.8). It is concluded that 5-HT induces relaxation of the canine rectum circular muscle through stimulation of a single population of smooth muscle 5-HT4 receptors. For the first time, a nonhuman species was shown to exhibit relaxant 5-HT4 receptors in the large intestine.

5-Methoxytryptamine↗

PubMed1998

Influence of selective VIP receptor agonists in the rat gastric fundus.

The receptor subtypes involved in the relaxant effect of vasoactive intestinal polypeptide (VIP) in the rat gastric fundus were investigated in vitro. The selective VIP2 receptor agonist [Ac-H1,E8,K12,Nle17,A19,D25,L26,K27,28,G29,30,++ +T31]VIP(cyclo21-25) (RO25-1553) induced a concentration-dependent relaxation (EC50 2.8 nM), while the selective VIP1 receptor agonist derived from growth hormone-releasing factor (GRF) [K15,R16,L27]VIP-(1-7)/GRF-(8-27) had no effect up to 1 microM. [R16] chicken secretin, a selective VIP1 receptor agonist, induced relaxation with a potency of 4.8 nM but its maximal effect was clearly lower than that of VIP, pituitary adenylate cyclase-activating peptide [PACAP-(1-27)] and RO25-1553. This effect was reproduced by porcine secretin (EC50 2.1 nM). It is concluded that the rat gastric fundus contains functional VIP2 receptors but not VIP1 receptors, and that specific secretin receptors are also present.

Animals↗

PubMed1998

Investigation of the influence of pregnancy on the role of nitric oxide in gastric emptying and non-adrenergic non-cholinergic relaxation in the rat.

The influence of pregnancy on the role of nitric oxide (NO) in gastric emptying and in non-adrenergic non-cholinergic (NANC) relaxation was studied in rats. The gastric emptying of a non-nutrient liquid solution and of polysterene beads was studied in non-pregnant (NP), 6 to 7 days pregnant (P7) and 18 to 20 days pregnant (P20) rats. Longitudinal muscle strips of the gastric fundus and circular muscle strips of the pylorus were isolated from NP and P20 rats and NANC relaxations were induced by electrical field stimulation. The gastric emptying of the liquid meal was significantly increased in P20 rats as compared to NP and P7 rats. In NP rats, NG-nitro-L-arginine methyl ester (L-NAME) dose-dependently (50-150 mg/kg i.p.) reduced the gastric liquid emptying; the inhibitory effect of 100 mg/kg L-NAME i.p. was prevented by 400 mg/kg i.p. L-arginine and was mimicked by 100 mg/kg NG-monomethyl-L-arginine (L-NMMA). The percentage inhibition of the liquid emptying by L-NAME did not differ between the 3 groups, except for the dose of 150 mg/kg i.p. where it was significantly lower in P20 rats. The gastric emptying of beads was 54% in NP, 36% in P7 and 69% in P20 rats but these values were not significantly different illustrating the great variability. The inhibitory effect of L-NAME (25 and 100 mg/kg i.p.) on the emptying of beads did not differ between the 3 groups. As evaluated in NP rats, the inhibitory effect of L-NAME on the gastric emptying of the beads was not prevented by L-arginine nor mimicked by L-NMMA. Electrical field stimulation in NANC conditions induced frequency-dependent relaxations in the fundus strips and relaxations followed by rebound contractions in the pyloric strips. These electrically induced NANC relaxations and their reduction by 3x 10(-4) M L-NAME were not different between NP and P20 rats. It can be concluded that no evidence for a regulatory role of NO in the gastric emptying of the beads was found, and that the nitrergic contribution to the gastric emptying of liquids and to the fundic and pyloric NANC relaxations was not influenced by pregnancy in rats.

Animals↗

PubMed1998

Effect of nitroglycerin and long-term electrical stimulation on nitrergic relaxation in the pig gastric fundus.

1. The effect of incubation with the nitric oxide (NO) donor nitroglycerin and of long-term electrical stimulation on relaxations induced by non-adrenergic, non-cholinergic nerve stimulation, exogenous NO, vasoactive intestinal polypeptide (VIP) and lemakalim was investigated in the pig gastric fundus. 2. In physiological salt solution containing 10(-6) M atropine and 4 x 10(-6) M guanethidine, electrical field stimulation (40 V, 0.1 ms, 0.5-8 Hz) for periods of 10 s at 5 min intervals (train stimulation) and administration of NO (2 x 10(-6)-10(-4) M) at 5 min intervals (NO boli) induced frequency- and concentration-dependent transient relaxations, respectively. Continuous electrical field stimulation with stepwise increase of the frequency (0.5-8 Hz, cumulative stimulation) induced frequency-dependent sustained relaxations. VIP (10(-7) M), lemakalim (10(-5) M) and an infusion of NO induced a sustained relaxation. 3. Pretreatment for 30 min with 5 x 10(-4) M nitroglycerin reduced the relaxations induced by train and cumulative stimulation, but also the relaxant responses to NO, both when given in boli or as an infusion. The relaxations to VIP and lemakalim were not influenced by pretreatment with nitroglycerin. 4. Long-term electrical stimulation at 4 Hz for 40 min induced a sustained relaxation of the tissues. Administration of 3 x 10(-4) M N(G)-nitro-L-arginine methyl ester after 10, 20 or 30 min reversed the relaxation to a similar extent (approximately 70%). Previous long-term electrical stimulation at 4 Hz for 30 min did not affect the responses to stimulation, NO and VIP. 5. These results illustrate that nitroglycerin can induce a postjunctional tolerance to nitrergic stimuli in the pig gastric fundus but evidence for a prejunctional inhibition of neuronal NO synthase by NO was not obtained.

Animals↗

PubMed1998

Influence of a selective guanylate cyclase inhibitor, and of the contraction level, on nitrergic relaxations in the gastric fundus.

1. The influence of the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) on non-adrenergic non-cholinergic (NANC) relaxations and the possible role of a nerve-derived hyperpolarizing factor in NANC relaxation were investigated in the rat gastric fundus. 2. ODQ (10(-6) and 10(-5) M) concentration-dependently inhibited the short-lasting relaxations by NO (2 x 10(-6) M-10(-4) M) administered as a bolus without influencing the relaxation by 3 x 10(-8) M isoprenaline. The relaxation by an infusion of NO was reduced to the same extent by 10(-6) and 10(-5) M ODQ. 3. The electrically induced short-lasting and sustained relaxations (40 V, 1 ms, 0.5-16 Hz, 10 s trains at 2 min interval or cumulative increase in the frequency every 2 min) in NANC conditions were inhibited to a similar extent by 10(-6) and 10(-5) M ODQ, and by the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 3 x 10(-4) M). 4. ODQ (10(-6) M) and L-NAME (3 x 10(-4) M), administered after 5, 10 or 20 min of long-term stimulation, reversed the relaxation to a similar extent (approximately 50% at 2 Hz and 20% at 8 Hz). 5. When the tissues were contracted to 40% of maximum by adapting the concentration of prostaglandin F2alpha (PGF2alpha), the inhibitory effect of 3 x 10(-4) M L-NAME on relaxations induced by train and cumulative stimulation was the same as when tissues were contracted with 3 x 10(-7) M PGF2alpha. 6. The findings of this study illustrate that the relaxation by exogenous and endogenous NO in the rat gastric fundus is due to activation of soluble guanylate cyclase. During long-term electrical stimulation, the partial contribution of NO to NANC relaxation is maintained but it is small at higher frequencies of stimulation. Evidence for the contribution of a nerve-derived hyperpolarizing factor to NANC relaxation was not obtained.

Animals↗

PubMed1998

Investigation of the interaction between cholinergic and nitrergic neurotransmission in the pig gastric fundus.

1. The interaction between the cholinergic and nitrergic innervation was investigated in circular muscle strips of the pig gastric fundus. 2. In physiological salt solution containing 4 x 10(-6) M guanethidine, electrical field stimulation (EFS; 40 V, 0.5 ms, 0.5-32 Hz, 10 s at 4 min intervals) induced small transient relaxations at 0.5-4 Hz, and large frequency-dependent contractions, sometimes followed by off-relaxations, at 8-32 Hz. 3. In the presence of L-NG-nitroarginine methyl ester (L-NAME; 3 x 10(-4) M) or physostigmine (10(-6) M), relaxations were reversed into contractions and contractions were enhanced. Physostigmine added to L-NAME further enhanced contractions, while addition of L-NAME to physostigmine had no additional effect. Off-relaxations were enhanced in the presence of L-NAME and physostigmine. L-NAME and physostigmine consistently increased basal tone. 4. Tissues contracted by 5-hydroxytryptamine or by acetylcholine responded to EFS in a similar way as in basal conditions and L-NAME reversed the relaxations at the lower stimulation frequencies into contractions and enhanced the contractions at the higher stimulation frequencies. 5. Off-relaxations in the presence of L-NAME were partially reduced by alpha-chymotrypsin (10 U ml(-1)). 6. In the absence of physostigmine, the concentration-response curve to exogenous acetylcholine was not influenced by L-NAME. 7. Contractions of the same amplitude induced by EFS at 4 Hz and by exogenous acetylcholine were either decreased or enhanced to the same extent by sodium nitroprusside (SNP; 10(-5) M), depending upon the degree of relaxation by SNP. 8. These experiments suggest that endogenous nitric oxide interferes with cholinergic neurotransmission in the pig gastric fundus by functional antagonism at the postjunctional level. The interaction is independent of the degree of contraction.

Acetylcholine↗

PubMed1997

Influence of endotoxin on contractility in the rat gastric fundus.

The influence of in vivo treatment with E. coli lipopolysaccharide endotoxin on the contractility of the rat gastric fundus was studied. Four h after lipopolysaccharide treatment (20 mg/kg i.p.), the contractile responses to prostaglandin F2alpha in longitudinal muscle strips from the gastric fundus were not different from those in control animals, while the well-known decreased response to noradrenaline in rings of the thoracic aorta was confirmed. Incubation of the tissues with L-arginine did not depress the response to prostaglandin F2alpha in fundus strips of lipopolysaccharide-treated rats. Twelve h after lipopolysaccharide treatment (6.7 mg/kg i.p.), the prostaglandin F2alpha-induced contractions were consistently depressed. The impairment of the prostaglandin F2alpha-induced responses by lipopolysaccharide treatment was not reversed by the nitric oxide synthase inhibitors NG-nitro-L-arginine (L-NNA, 10(-4) M), NG-nitro-L-arginine methyl ester (L-NAME, 3 x 10(-4) M), aminoguanidine (10(-4) M) and L-N6-l-iminoethyl-lysine (L-NIL, 10(-4) M) nor by the cyclooxygenase inhibitor indomethacin (10(-5) M). The impairment was prevented by pretreating the animals with dexamethasone (5 mg/kg i.p.), which had no effect per se on the contractile response to prostaglandin F2alpha. Lipopolysaccharide treatment did not influence the contractile responses to KCl and serotonin. The nonadrenergic noncholinergic relaxant responses to transmural electrical stimulation were not influenced 4 h after lipopolysaccharide treatment but were moderately reduced after 12 h. The results illustrate that the selective impairment of prostaglandin F2alpha-induced contractions in the rat gastric fundus by lipopolysaccharide treatment is not mediated via generation of nitric oxide; downregulation of the prostaglandin F2alpha-receptor by lipopolysaccharide treatment might be involved.

Abortifacient Agents, Nonsteroidal↗

PubMed1997

Role of 5-hydroxytryptamine and mast cells in the tachykinin-induced contraction of rat trachea in vitro.

The in vivo bronchoconstrictor effect of tachykinins in Fisher 344 rats is accompanied by release into the airways of 5-hydroxytryptamine (5-HT). 5-HT is possibly derived from mast cells. In the present study the presumed mast cell-tachykinin interaction was studied in isolated trachea from Fisher 344 rats. Contractions induced by neurokinin A were largely reduced by the 5-HT antagonist methysergide, partially reduced by atropine, but not affected by hexamethonium or tetrodotoxin. Methysergide also inhibited the contractions induced by substance P, the tachykinin NK1 receptor agonist Ac[Arg6, Sar9, Met(O2)11]substance P-(6-11) and the mast cell depleting compound 48/80. Methysergide had no effect on contractions induced by carbachol or electrical field stimulation. Atropine significantly reduced contractions to 5-HT and completely inhibited contractions induced by electrical field stimulation. Histamine had no contractile effect. In vivo pretreatment with compound 48/80 significantly reduced the in vitro contractions to neurokinin A. Contractions to capsaicin were inhibited by methysergide and the tachykinin NK1 receptor antagonist (+/-)-RP67580 ((3alphaR,7alphaR)-(7,7-diphenyl-2-(1-imino-2-(2-methoxyp henylethyl)-perhydraisoinotol-4-one))). Substance P and neurokinin A caused 5-HT release in the organ bath, in a concentration- and time-dependent way. Atropine did not affect 5-HT release. Morphometric analysis showed that substance P and neurokinin A, but not carbachol, caused a significant increase in the number of degranulating mast cells in the muscular/submuscular region. In conclusion, tachykinins contract Fisher 344 rat trachea by releasing 5-HT from mast cells, an effect mediated by a tachykinin NK1 receptor.

Animals↗

PubMed1997

Influence of different classes of NO synthase inhibitors in the pig gastric fundus.

The inhibitory potency of different classes of nitric oxide synthase (NOS) inhibitors (amino acid-based substances, guanidines, isothioureas, imidazoles and indazoles) versus peripheral neuronal NOS in the pig gastric fundus was investigated by studying their influence on electrically induced relaxations in non-adrenergic noncholinergic conditions. Circular muscle strips were mounted for isotonic registration in the presence of atropine and guanethidine, and tone was raised with 5-hydroxytryptamine. Electrical field stimulation (40 V, 0.1 ms, 4 Hz, 10 s) induced short-lasting relaxations. The inhibitory effect of 1-phenylimidazole could not be evaluated because it nearly abolished the 5-hydroxytryptamine-induced tone of the tissues. 7-Nitroindazole, imidazole, 2-iminobiotin and aminoguanidine did not inhibit the electrically induced relaxations, while the other 9 substances tested were able to do so. The influence of the incubation period was tested by studying the inhibitory effect after incubation for 10 up to 60 min. For N(G)-nitro-L-arginine methyl ester (L-NAME), N(G)-nitro-L-arginine (L-NNA), L-N5-(1-iminoethyl)-ornithine (L-NIO), L-N6-(1-iminoethyl)-lysine (L-NIL), S-methyl-L-thiocitrulline and S-isopropyl isothiourea there was a moderate increase in the inhibitory effect up to 30 min of incubation so that they were incubated for 30 min to study their inhibitory potency. For L-thiocitrulline, S-methyl isothiourea and S-ethyl isothiourea, an incubation period of 60 min was used. The 9 substances concentration-dependently inhibited the electrically induced relaxations with a maximal inhibitory effect of approximately 80% except for S-methyl isothiourea (Emax of 53%). The overall order of potency was: S-isopropyl isothiourea > S-ethyl isothiourea > or = S-methylL-thiocitrulline > or = L-NNA > L-NIO > L-NAME > S-methyl isothiourea > L-thiocitrulline > L-NIL. While the potency for S-isopropyl isothiourea (EC50: 3.1 x 10(-5) M, n = 6) to S-methyl isothiourea (EC50: 11.5 x 10(-5) M, n = 5) was in the same range, the potency of L-thiocitrulline and L-NIL was clearly lower. This study showed several compounds to be potent inhibitors of peripheral neuronal NOS in the pig gastric fundus while some compounds, that were reported to inhibit brain neuronal NOS were not effective. The EC50 values found for the effective substrates in this functional study may be a guideline for the concentrations required to evaluate the role of NO in NANC neurotransmission in gastrointestinal smooth muscle preparations.

Animals↗

PubMed1997

Evaluation of an electrochemical microprobe for direct NO measurement in the rat gastric fundus.

Nitric oxide (NO) is an inhibitory nonadrenergic, noncholinergic (NANC) neurotransmitter in the rat gastric fundus and is released upon electrical or pharmacological stimulation of the inhibitory NANC neurons. In this study, it was attempted to measure the release of NO from the rat gastric fundus upon electrical stimulation or administration of nicotine directly via an electrochemical probe (ISO-NO). The system was evaluated by adding exogenous NO. Addition of exogenous NO induced concentration-dependent relaxation of the rat gastric fundus and an increase in the ISO-NO probe baseline current. The concentration of NO detected by the ISO-NO probe was lower than the concentration of NO administered. When no tissue was present, higher concentrations of NO were detected than in the presence of a tissue. In the absence of 95% O2/5% CO2 the concentration of NO detected was highest. Electrical stimulation induced relaxations of the rat gastric fundus which were reduced by NG-nitro-L-arginine methylester (L-NAME). An increase in the ISO-NO probe baseline current was also observed, but this was duc to nonspecific effects as the response also occurred without a tissue present and was not sensitive to L-NAME. Nicotine induced relaxations, which were reduced by L-NAME, but the ISO-NO probe baseline current remained unaltered, even in the presence of L-arginine plus superoxide dismutase. It can be concluded that it is not possible to detect directly the NO release from the rat gastric fundus upon electrical or pharmacological stimulation of the NANC neurons with the ISO-NO probe.

Animals↗