Guinea Pig Ileum Preparations Research Articles

VB20B7, a novel 5-HT-ergic agent with gastrokinetic activity. I. Interaction with 5-HT3 and 5-HT4 receptors.

This study describes the in-vitro interaction of the gastrokinetic agent 2[1-(4-piperonyl)piperazinyl]benzothiazole (VB20B7) with the 5-hydroxytryptamine 5-HT3 and 5-HT4 receptor subtypes, using functional as well as radioligand binding studies. The benzamide derivative cisapride was used as a comparison. In radioligand binding assays VB20B7 showed, like cisapride, a weak affinity at 5-HT3 receptors from rat cerebral cortex. The new compound lacked any affinity at other 5-HT receptors or at dopaminergic D2 receptors, whereas cisapride showed high affinity for the 5-HT4 receptors from guinea-pig hippocampus and moderate affinity at dopaminergic D2 receptors. In the non-stimulated guinea-pig ileum, the concentration-response curves to the specific 5-HT3 agonist 2-Me-5-HT and to 5-HT were shifted to the right by VB20B7. In the rat oesophagus tunica muscularis mucosae preparation (TMM), VB20B7 was evaluated for its activity at 5-HT4 receptors. VB20B7 behaved as a 5-HT4 receptor agonist, inducing a concentration-dependent relaxation of the preparation precontracted with carbachol. In this preparation, VB20B7 and cisapride were able to stimulate adenylate cyclase activity, an effect probably mediated through activation of 5-HT4 receptors, as can be inferred from the blockade by the 5-HT4 antagonist, tropisetron, of the enhanced cAMP formation. However, consistent with the lack of affinity at central 5-HT4 receptors, VB20B7 did not stimulate cAMP formation in guinea-pig hippocampal slices. VB20B7 also caused an increase in the twitch response of the transmurally stimulated guinea-pig ileum, although at a concentration higher than cisapride. This effect was blocked by desensitization of the 5-HT4 receptor with 5-MeOT and also by the 5-HT4 receptor antagonist tropisetron. Both VB20B7 and cisapride increased the K(+)-evoked acetylcholine release in this preparation. The results show that VB20B7 possesses affinity for 5-HT4 receptors located in the rat TMM and guinea-pig ileum preparations, but is devoid of affinity at central 5-HT4 receptors. In addition, VB20B7 shows low to moderate affinity at both central and peripheral (enteric) 5-HT3 receptors. The interaction of VB20B7 with the peripheral 5-HT4 and 5-HT3 receptors may be relevant for the gastrokinetic effects of the new compound.

Depressant effects of hypoxia and hypoglycaemia on neuro-effector transmission of guinea-pig intestine studied in vitro with a pharmacological model.

1. Since intermittent ischaemia may play an important role in the aetiology of Inflammatory Bowel Disease, particularly Crohn's Disease, a pharmacological model of neuronal ischaemia was applied to guinea-pig isolated intestinal preparations to mimic the acute effects of reduced blood flow on intestinal motility. 2. Neuro-effector transmission and smooth muscle performance were examined in myenteric plexus-longitudinal muscle preparations of guinea-pig ileum exposed to sodium cyanide (NaCN), in order to inhibit oxidative phosphorylation, or to iodoacetic acid (IAA), to block glycolysis. Comparisons were made with the effects due to simple deprivation of oxygen or glucose. 3. Depression of cholinergic neuro-effector transmission induced by hypoxia or NaCN (effective concentration range 0.1-3 mM), given as separate treatments, singly or repetitively over 60-90 min, were apparent within 30 s and were reversible. The maximum inhibition was 90% and the IC50 for NaCN was 0.3 mM. A conspicuous component of these inhibitions was prejunctional. 4. Non-cholinergic neuro-effector contractions were inhibited by up to 90% by anoxia or NaCN but recovery was incomplete and slower than with cholinergic contractions. 5. Glucose-free solutions also caused a reversible failure of cholinergic neuro-effector transmission but of slower onset. In contrast, IAA (0.06-1 mM) abolished contractions irreversibly, apparently by a direct depressant effect on smooth muscle contraction. Unlike NaCN, IAA caused an initial potentiation of electrically-induced contractions, partly by a prejunctional potentiation of cholinergic neuro-effector transmission. 6. It is concluded that a disruption of intestinal activity in pathological conditions associated with intestinal ischaemia may result from disturbances in the function of enteric neurones.

Biologic activities of iodinated analogues of Tyr 0-bradykinin and bradykinin-Ile 10-Tyr 11 assessed in the rat uterus and the guinea pig ileum

1. 1. The biological activity of bradykinin (BK) and analogues containing Tyr in extended N- or C-terminal portions of the molecule, as well as that of their iodinated products, was compared in isolated rat uterus and guinea pig ileum preparations. 2. 2. BK-Tyr 10 and BK-Ile 10-Tyr 11 were obtained by solid phase synthesis employing fmoc chemistry. 3. 3. Iodination of BK-Ile 10-Tyr 11 and Tyr 10-BK was performed using iodobeads R, and the products were purified by reverse-phase HPLC. 4. 4. The relative potency (RP) of noniodinated analogues in the uterus was: Tyr 0-BK (1.3)=BK (1.0)>BK-Ile 10-Tyr 11(0.45)>>BK-Tyr 10 (0.02) and BK (1.0)>BK-Ile 10-Tyr 11 (0.25)=Tyr 0-BK (0.22)>>BK-Tyr 10(0.002). The RP of mono-iodo (MI) and di-iodo (Dl) products was: BK (1.0)>IBK-Ile 11-Tyr 11 (0.63)=DI-Tyr 0-BK (0.63)>MI-Tyr 0-BK (0.46)=MI-BK-Ile 10-Tyr 10 (0.40). 5. 5. The RP of noniodinated analogues in the guinea pig ileum was: BK (1.0)>MI-Tyr 0-BK (0.39)>MI-BK-Ile 10-Tyr 11 (0.17)=DI-Tyr 0-BK (0.16)=DI-BK-Ile 10-Tyr 11(0.13). 6. 6. Differences in RP of 8–10 fold for Tyr 0-BK or BK-Tyr 10 and 2-fold for BK-Ile 10-Tyr 11 were observed between the two preparations used, indicating possible receptor differences. 7. 7. Iodination caused a reduction in the RP of the analogues in both preparations. 8. 8. In the rat uterus, the changes in the RP of the Tyr 0-BK analogues were more evident than those observed with the iodinated analogues of BK-Ile 10-Tyr 11, indicating that iodination causes different changes in RP, according to the localization of the Tyr in the molecule. 9. 9. The data support the idea that iodinated analogues of BK-Ile 10-Tyr 11, with intact N-terminal portion, may be as useful as iodinated analogues of Tyr- 0-BK for the study of BK receptors.

Interaction between opioid and muscarinic receptors in the guinea-pig ileum preparation: a mathematical model.

Fentanyl and pethidine are opioid agonists and muscarinic antagonists in the guinea-pig ileum preparation. In this preparation an opioid agonist reduces the release of acetylcholine. Therefore an opiate may influence the potency of an anticholinergic drug. A mathematical model was developed to characterize this putative interaction between opioid and muscarinic receptors. The model is based on the assumption that the drugs interact with the receptors in a competitive manner according to the law-of-mass action. Concentration-response experiments were performed in the guinea-pig ileum preparation to test the model. The mathematical model describes the concentration-response curves very well and estimates the IC50 values for the two components with good precision. The study shows that an opioid agonist can potentiate the effect of an anticholinergic drug substantially. This is interesting with regard to the central anticholinergic syndrome. The conclusion is that the model describes the interaction adequately.

Potential irreversible ligands for opioid receptors. Cinnamoyl derivatives of beta-naltrexamine.

Cinnamoyl derivatives of beta-naltrexamine (beta-NTA) have been prepared and evaluated as potential irreversible opioid antagonists. In receptor binding assays, isolated tissue preparations and mouse antinociception assays the p-methylcinnamoyl derivative BU42 was similar to the standard opioid ligand beta-funaltrexamine (beta-FNA). The main features were reversible kappa agonism and irreversible mu antagonism. Surprisingly the p-chlorocinnamoyl derivative BU59 showed only modest competitive antagonist activity in-vivo despite appearing to bind irreversibly to mu receptors in the guinea-pig ileum (GPI) preparation. BU60, the dihydrocinnamoyl analogue of BU59, like BU59 displayed reversible kappa agonism in GPI but in mouse antinociception assays its agonism was mediated by mu and delta receptors rather than kappa. The surprising changes of profile attributable to substitution in the aromatic ring of the cinnamoylamido group in this small series suggests that a larger range of substituted cinnamoylamido derivatives should be studied to further elucidate the effects of Michael acceptor activity and other factors.

N-Cubylmethyl substituted morphinoids as novel narcotic antagonists

N-Cubylmethylnormorphine (1) and N-cubylmethylnoroxymorphone (2) have been synthesized and found to be more potent ligands at the μ and κ opioid receptors than morphine and oxymorphone respectively. In the guinea-pig ileum preparation, compounds 1 and 2 were characterized as opioid μ antagonists (Ke = 68 and 16 nM, respectively). Compound 2 also showed effective κ-antagonism (Ke = 22 nM). The narcotic antagonism activity of 1 has been confirmed by in vivo assays.

Interaction of the chiral forms of ketamine with opioid, phencyclidine, sigma and muscarinic receptors.

In order to elucidate the mechanisms of action of ketamine, we have investigated the binding of the chiral forms of ketamine to opioid (mu, delta and kappa), phencyclidine, sigma and muscarinic receptors and we have performed detailed concentration-response experiments in the guinea-pig ileum preparation. The affinity ratios for the chiral forms at phencyclidine, mu and kappa receptors correlated with the potency ratio of the chiral forms in the ischaemic pain test found previously. The affinities were highest for phencyclidine receptors. The affinities for muscarinic receptors were lower than for phencyclidine receptors by a factor of about 10-20. The concentration-response experiments revealed one opioid (naloxone sensitive) and one non-opioid component. The two component are very close, which explains why other authors have reported that naloxone antagonizes the ketamine effect only partly. The concentrations of naloxone necessary to shift the opioid part of the curves indicate that ketamine is a kappa agonist in the guinea-pig ileum preparation. We conclude that the analgesic effect of ketamine in humans is most probably mediated via phencyclidine receptors, although a kappa effect can not be excluded. Binding to kappa and muscarinic receptors may contribute to the psychotomimetic side effects seen during recovery from ketamine anaesthesia.

σ2 Site-mediated inhibition of electrically evoked guinea pig ileum longitudinal muscle/myenteric plexus contractions

Functional and binding studies were performed in order to characterize the relative efficacy and affinity of a number of compounds that bind to σ sites. The ability of σ site ligands to inhibit electrically evoked contraction of the guinea pig ileum longitudinal muscle/myenteric plexus preparation was compared to the affinities of these compounds for σ 1 sites (assessed by displacement of [ 3H](+)-pentazocine) and σ 2 sites (assessed by displacement of [ 3H]1,3-di- o-tolylguanidine (DTG) in the presence of 5 μM dextromethorphan). It was shown that the rank order of potencies for suppression of electrically evoked contractions of guinea pig ileum perfectly matched the rank order of affinities of these compounds for the σ 2 binding site, while correlating poorly with the σ 1 binding site. In addition, no significant correlations were found between the efficacy of the tested compounds to inhibit contraction of the guinea pig ileum preparation and previously reported affinities for muscarinic, dopamine D 2 or MK-801 binding sites. Thus, the present study represents the first functional bioassay selectively sensitive to agents interacting with the σ 2 receptor subtype binding site, and provides a means with which to further elucidate the functional role of σ 2 sites.

Competitive and selective antagonistic action of NPC 17731 on bradykinin-induced relaxant and contractile responses of the guinea pig isolated ileum

1. 1. The aim of the present study was to evaluate, by means of functional studies, the effect of the newly developed bradykinin (BK) receptor antagonist NPC 17731, D-Arg 0, Arg 1-Pro 2-Hyp 3-Gly 4-Phe 5-Ser 6-[DHype (transpropyl) 7]-Oic 8-Arg 9, against BK-mediated biphasic response in the guinea pig ileum “in vitro” (circular and longitudinal layers). 2. 2. In the circular muscle, NPC 17731 (0.1–1000 nM) dose dependently and reversibly antagonized, with similar potency, both the contractile and relaxant responses caused by BK, as well as BK-induced contraction in longitudinal smooth muscle with IC 50 of 23, 29 and 37 nM, respectively. 3. 3. NPC 17731 (10–300 nM) also caused a concentration-dependent displacement to the right of BK-induced contraction and relaxant responses in the circular smooth muscle of guinea pig ileum, without changing BK maximal response. Schild plot analyses were linear (correlation close to 1), yielding pKb values of 8.89 ± 0.19 and 8.73 ± 0.18, respectively, and slopes not significantly different from unity, providing evidence that NPC 17731 acts as a pure B 2 competitive receptor antagonist against both BK responses. 4. 4. Similarly, NPC 17731 (3-100 nM) antagonized, in a graded and competitive manner, BK-induced contraction in longitudinal muscle from guinea pig ileum with a slope not different from unity, yielding a pKb value of 8.62 ± 0.13. 5. 5. These results indicate that the new B 2 BK receptor antagonist NPC 17731 antagonizes, with high affinity and with similar potency through simple competitive and selective mechanisms, BK receptor-mediating contraction or relaxant responses in circular and longitudinal smooth muscles from guinea pig ileum. In addition, these results also suggest that although BK-induced contraction or relaxation responses in guinea pig ileum are coupled to distinct second messengers, NPC 17731 interacts with a homogeneous population of B 2 receptors in both guinea pig ileum preparations.

δ Opioidmimetic Antagonists: Prototypes for Designing a New Generation of Ultraselective Opioid Peptides

Tyr-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) and Tyr-Tic-Ala were the first peptides with delta opioid antagonist activity lacking Phe, considered essential for opioid activity based on the N-terminal tripeptide sequence (Tyr-D-Xaa-Phe) of amphibian skin opioids. Analogs were then designed to restrain the rotational flexibility of Tyr by the substitution of 2,6-dimethyl-L-tyrosine (Dmt). Tyr and Dmt peptides were synthesized by solid phase and solution methods using Fmoc technology or condensing Boc-Dmt-OH or Boc-Tyr(But)-OH with H-L-Tic-OBut or H-D-Tic-OBut, respectively. Peptides were purified (> 99%) by HPLC and characteristics determined by 1H-NMR, FAB-MS, melting point, TLC, and amino acid analyses. H-Dmt-Tic-OH had high affinity (Ki delta = 0.022 nM) and extraordinary selectivity (Ki mu/Ki delta = 150,000); H-Dmt-Tic-Ala-OH had a Ki delta = 0.29 nM and delta selectivity = 20,000. Affinity and selectivity increased 8700- and 1000-fold relative to H-Tyr-Tic-OH, respectively. H-Dmt-Tic-OH and H-Dmt-Tic-NH2 fitted one-site receptor binding models (eta = 0.939-0.987), while H-Dmt-Tic-ol, H-Dmt-Tic-Ala-OH and H-Dmt-Tic-Ala-NH2 best fitted two-site models (eta = 0.708-0.801, F 18.9-26.0, p < 0.0001). Amidation increased mu affinity by 10- to 100-fold and acted synergistically with D-Tic2 to reverse selectivity (delta-->mu). Dmt-Tic di- and tripeptides exhibited delta antagonist bioactivity (Ke = 4-66 nM) with mouse vas deferens and lacked agonist mu activity (> 10 microM) in guinea-pig ileum preparations. Dmt-Tic analogs weakly interacted with kappa receptors in the 1 to > 20 microM range. Dmt-Tic opioidmimetic peptides represent a highly potent class of opioid peptide antagonists with greater potency than the nonopioid delta antagonist naltrindole and have potential application as clinical and therapeutic compounds.

Putative mechanism for guinea pig ileum contraction by N-formyl peptides. a comparative study of n-formyl and N-acetyl peptides with the N-terminal sequence of the calpain small subunit

N-formyl and N-acetyl peptides with the N-terminal sequence of the calpain small subunit were prepared and their spasmogenic activity was examined using guinea pig ileum preparations. Sections of ileum were found to contract in the presence of all N-formyl peptides used (tri- to nonapeptides and tridecapeptide) but failed to contract with N-acetyl peptides, although both N-formyl and N-acetyl peptides have chemotactic activity, indicating that spasmogenic activity and chemotactic activity involve different mechanisms. A formyl peptide antagonist, Boc-Phe-Leu-Phe-Leu-Phe, suppressed contraction by formyl peptides whereas a histamine antagonist, diphenhydramine. suppressed contraction by formyl peptides as well as by histamine. In addition, formy] peptide-induced contractions were noted after an approximately 20-sec time lag, and their profiles were bell-shaped and roughly symmetrical. On the other hand, histamine- and acetylcholine-induced contractions exhibited a much shorter time lag. These data led us to conclude that contraction induced by formyl peptides may not occur as a direct response but may be due to the histamine released from mast cells present in the tissues of the small intestine.

The serotonin 5-HT4 receptor. 2. Structure-activity studies of the indole carbazimidamide class of agonists.

A number of substituted indole carbazimidamides were prepared and evaluated as 5-HT4 receptor agonists by using the isolated field-stimulated guinea pig ileum preparation. Their selectivity for the 5-HT4 receptor was established by examining their affinity for other 5-HT receptors using radioligand-binding techniques. Several selective and highly potent full as well as partial agonists emerged from this study. For example, 1b,d were found to be the most potent, full 5-HT4 receptor agonist described so far (EC50 = 0.5 and 0.8 nM, respectively), being 6 and 4 times more potent than serotonin itself. On the other hand, 5b and 1h appeared as partial 5-HT4 receptor agonists in the nonstimulated guinea pig ileum preparation with potencies, evaluated against serotonin action, respectively similar (5b, Ki = 12 nM) to and 300-fold higher (1h, Ki = 0.04 nM) than serotonin.

The serotonin 5-HT4 receptor. 1. Design of a new class of agonists and receptor map of the agonist recognition site.

The design and synthesis of a new class of potent and selective 5-HT4 receptor agonists containing an indole nucleus linked to a carbazimidamide are presented. A conformational study of the 5-HT4 receptor agonists serotonin and zacopride led to the identification of an initial pharmacophore and to the definition of a three-dimensional map of the 5-HT4 agonist recognition site. 1, a representative member of our new class of 5-HT4 receptor agonists, incorporates all reference structural features and matched perfectly with these models. 1 is a highly potent, full agonist at 5-HT4 receptors present in the isolated electrically stimulated guinea pig ileum preparation, with a pD2 value of 8.8, displaying selectivity (ranging from 40- to over 10,000-fold) versus other members of the serotonin receptor family.

The in vitro pharmacological characterization of naloxone benzoylhydrazone

On the basis of its in vivo activity and binding affinity, naloxone benzoylhydrazone has been characterized as a κ 3-opioid receptor agonist and a μ-opioid receptor antagonist. This paper continues its pharmacological characterization with the help of isolated tissue preparations. Naloxone benzoylhydrazone was found to have partial agonist activity in the guinea pig ileum longitudinal muscle/myenteric plexus preparation. As an antagonist, naloxone benzoylhydrazone is similar to naloxone, with pA 2 values of 8.8, 7.8, and 7.8 for μ-, δ-, and κ 1-opioid receptors, respectively. Its agonist activity in the guinea pig ileum preparation was not influenced by β-funaltrexamine treatment but was reversed by the selective κ-opioid receptor antagonist nor-binaltorphimine and by the irreversible κ 1-opioid receptor blocker UPHIT (1 S,2 S)- trans-2-isothiocyanato-4,5-dichloro- N-methyl- N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide. The presence of κ 3-opioid receptors could not be demonstrated by [ 3H]naloxone benzoylhydrazone binding in the guinea pig ileum longitudinal muscle/myenteric plexus preparation. From these studies it is concluded that the partial agonist activity of naloxone benzoylhydrazone in this bioassay is probably due to the activation of the κ 1-opioid receptors.

Effects of methylnaltrexone on morphine-induced inhibition of contraction in isolated guinea-pig ileum and human intestine

We investigated the effects of methylnaltrexone on morphine-induced inhibition of smooth muscle-strip contraction in isolated guinea-pig ileum and human small intestine. The longitudinal muscle-strip was immersed in a temperature-controlled (37°C) bath containing a physiological solution of 95% O 2 and 5% CO 2 with pH 7.4. Muscle contraction was elicited by transmural electrical stimulation with a pulse duration of 0.5 ms at frequencies of 1–50 Hz for 5–10 s at 1–3-min intervals. Muscle contraction was blocked by tetrodotoxin or atropine in both preparations. When methylnaltrexone was applied to the bath, the force produced by muscle contraction was enhanced up to approximately 30%. Stimulation-elicited muscle contraction was inhibited by morphine, which decreased the force of contraction 42 ± 9.5% (S.D.) in the human intestine preparation and 35 ± 8.6% in guinea-pig ileum at the inhibitory concentration 70% (IC 70). Methylnaltrexone effectively antagonized the effects of morphine-induced inhibition of muscle-strip contraction. In the guinea-pig ileum preparation, methylnaltrexone at 30, 100 and 300 nM blocked 25 ± 10.5%, 74 ± 7.2% and 89 ± 9.9% of morphine-induced (300 nM) inhibition, respectively. In the human intestine preparation, methylnaltrexone at the same concentrations blocked 57 ± 10.9%, 74 ± 12.9% and 92 ± 7.2% of morphine-induced (100 nM) inhibition, respectively. The relative ratio of methylnaltrexone to morphine was higher in human intestine (1:1) than in the guinea-pig ileum preparation (1:3). These data provide preliminary information for clinical studies to evaluate the efficacy of methylnaltrexone in preventing or reducing morphine-induced antimotility and antitransit actions.

Acute withdrawal after bremazocine and the interaction between μ‐ and κ‐opioid receptors in isolated gut tissues

1. This study was undertaken to investigate whether, after a brief exposure of guinea-pig isolated ileum and rabbit jejunum to bremazocine, a kappa-opioid agonist also possessing antagonist activity at mu-opioid receptors, the addition of opioid antagonists produced withdrawal contractures. Our aim was to verify in these tissues the existence of an interaction between the mu- and kappa-opioid systems. 2. In guinea-pig ileum preparations previously exposed for 5 min to bremazocine at 5.7 x 10(-7) M and 5.7 x 10(-8) M, naloxone (5 x 10(-7) M) elicited no response whereas in tissues exposed to a lower bremazocine concentration (5.7 x 10(-9) M), naloxone (5 x 10(-7) M) and the selective kappa-opioid antagonist, nor-binaltorphimine (3.4 x 10(-8) M) both produced a strong contracture. 3. Bremazocine (5.7 x 10(-7) M) administered to guinea-pig isolated ileum, previously exposed for 5 min to morphine (10(-7) M), induced a withdrawal contracture. In contrast, lower bremazocine concentrations (1.4 and 7.1 x 10(-8) M) did not elicit a withdrawal contracture. 4. Naloxone (5 x 10(-7) M), added to the bath after a 5 min exposure of guinea-pig ileum to morphine (10(-7) M), elicited the characteristic withdrawal contracture. Bremazocine (1.4-7.1 x 10(-8) M) added 1 min before naloxone (5 x 10(-7) M) inhibited the naloxone withdrawal contracture in a dose-related way whereas naloxone 5 x 10(-8) M added 1 min before naloxone 5 x 10(-7) M, did not affect the withdrawal response. 5. In the rabbit jejunum, bremazocine (1.4-7.1 x 10-8 M) caused a decrease in amplitude in the spontaneous tissue activity. In tissues exposed to these bremazocine concentrations, naloxone(5 x 10-7 M) elicited a marked contracture. A similar contracture occurred when nor-binaltorphimine(3.4 x 10-8 M) was added in place of naloxone. These effects were dose-related to the bremazocine concentration. The specific K-agonist, U-50,488H (5 x 10-8 M), elicited the same effects as bremazocine.6. These findings show that stimulation of K-opioid receptors induces a state of dependence that is not prevented by blocking the pi-opioid system. The observation that low bremazocine concentrations inhibit the morphine-induced withdrawal contractures, indicates an interaction between the micro- and K-opioid system in guinea-pig isolated ileum, similar to that observed in the whole animal.

Isolation of cholinergic active ingredients in aqueous extracts of Mareya micrantha using the longitudinal muscle of isolated guinea-pig ileum as a pharmacological activity marker

In our attempt to isolate the pharmacologically active ingredients in the aqueous extracts of Mareya micrantha, we have selected the contractions of the longitudinal muscle of the isolated guinea-pig ileum preparation as a pharmacological marker to monitor retention of pharmacological activity during the chromatographic separation. The aqueous extracts of Mareya micrantha elicited concentration-dependent contractions of the preparation. The maximum response elicited by the aqueous extract was 50% of the maximum response elicited by the maximum dose of acetylcholine (ACh), 10 −7 M. Mepenzolate (10 −8–10 −5 M), a specific muscarinic receptor antagonist, similarly antagonized contractions elicited by the aqueous extracts suggesting that the cholinergic ingredient(s) in the extract are acting at the muscarinic receptors of the preparation. Fraction 2–4, which was separated from the aqueous extracts by Sephadex gel chromatography, dose-dependently elicited contractions of the preparation. The maximum response was 80% of the maximum response elicited by the maximum dose of ACh suggesting that separation has enhanced the cholinergic activity of the content in the extract.

Effects of Aminoglycoside Antibiotics on Cholinergic Autonomic Nervous Transmission

The effects of eight aminoglycoside antibiotics, gentamicin, neomycin B, ribostamycin, dibekacin B, kanamycin A, streptomycin, tobramycin and amikacin, and two non-aminoglycoside antibiotics, tetracycline and ampicillin, on cholinergic autonomic nervous transmission were studied using isolated guinea-pig ileum preparation. The aminoglycoside antibiotics blocked the transmurally elicited twitches of the ileum in a concentration-dependent manner. The blocking effect of aminoglycosides was biphasic, i.e., an initial reduction followed by a spontaneous partial recovery. Dibekacin was the most potent parasympathetic inhibitor, followed by neomycin B, tetracycline, gentamicin, streptomycin, kanamycin A, tobramycin, ribostamycin, and amikacin. Ampicillin had no blocking effect. The tested antibiotics did not affect acetylcholine (ACh)-induced contraction of the ileum, except for high concentrations of neomycin B, gentamicin, and streptomycin. The three antibiotics shifted the dose-response curves for ACh to the right without affecting the maximal contraction. Naloxon, yohimbine, hexamethonium and choline chloride failed to eliminate the blocking effect of the antibiotics on twitches of the ileum induced by transmural stimulation. However, increase of the extracellular Ca ion concentration virtually abolished the blockade. Dibekacin blocked the evoked but not the spontaneous release of ACh and shifted the dose-response curve of CaCl2-dependent transmurally elicited contractions of the ileum to the right. These results suggest that the site for the block of aminoglycosides is mainly the cholinergic nerve terminal, where they reduce the available Ca ions required for the release of ACh.

Spontaneous Tone in Different Types of Longitudinal Muscle Preparations of Guinea Pig Ileum

Spontaneous tone of longitudinal muscle in guinea pig ileum was investigated in three types of preparations, intact segment preparation, segment preparation deprived of the mucosal layer and longitudinal strip preparation. Tone was defined as the sustained contraction that was lost by 10−7 M atropine in the isotonically recorded manner. The magnitudes of tone were constant for at least 3 hr in the two types of segment preparations. Contractions in response to 10−6 M acetylcholine, which were induced 9 times with an interval of 20 min between each induction, were almost identical throughout the period. In the longitudinal strip preparation, on the other hand, the tone gradually decayed and was eventually lost, while the amplitudes of acetylcholine-induced contractions were reciprocally increased. The tone in the intact segment preparation was reduced to 19% of the control by tetrodotoxin (3 × 10−7 M), to 51% by indomethacin (3 × 10−6 M) and to 26% by N6-cyclopentyladenosine (10−7 M), but was not affected by AA-861 (3 × 10−6 M) or CP-96,345 (3 × 10−7 M). In the three types of preparations, the dose-response curves for acetylcholine were alike with similar EC50s. These results suggest that the tone of longitudinal muscle was mainly induced due to neural activity in the myenteric plexus of guinea pig ileum and that sensitivity to acetylcholine was not affected by the neural activity.

Antagonistic activities of N-3389, a newly synthetized diazabicyclo derivative, at 5-HT 3 and 5-HT 4 receptors

The antagonistic activities of compound N-3389 ( endo-3,9-dimethyl-3,9-diazabicyclo[3,3,1]non-7-yl 1 H-indazole-3- carboxamide dihydrochlo ride) at 5-HT 3 and 5-HT 4 receptors were examined using in vitro and in vivo assays. N-3389 showed potent 5-HT 3 receptor antagonistic activities in a radioligand binding assay (p K i = 8.77), against 2-methyl-5-HT (2-Me-5-HT)-induced bradycardia in rats (ED 50 = 0.73 μg/kg i.v., 38 μg/kg p.o.) and against 2-Me-5-HT-induced contraction in longitudinal muscle myenteric plexus preparations of guinea-pig ileum (IC 50 = 3.2 × 10 −8 M). As preliminary to investigating the effect of N-3389 on 5-HT 4 receptors, we examined the contraction induced by 5-HT in guinea-pig ileum preparations. We confirmed that 5-HT (10 −8 − 10 −5 M) induced biphasic contractions in the preparations.Furthermore, 5-HT 3 receptor antagonism inhibited the late phase of the contraction induced by high concentrations of 5-HT (3 × 10 −6 − 10 −5 M), whereas 5-HT 4 receptor antagonism inhibited the early phase of the contraction induced by low concentrations of 5-HT (10 −8 − 10 −6 M). N-3389 (10 −7 − 10 −5 M) inhibited both phases of contraction induced by 5-HT. In addition, N-3389 (3 × 10 −7 −3 × 10 −6 M) was found to inhibit the increase of electrically stimulated twitch responses induced by 5-HT (10 −8 M) in longitudinal muscle myenteric plexus preparation of the guinea-pig ileum. These results suggest that N-3389 acts as a 5-HT 3 and 5-HT 4 receptor antagonist.