Guinea-pig Ileal Preparations Research Articles

Neurophysiological mechanisms of bradykinin-evoked mucosal chloride secretion in guinea pig small intestine

To investigate the mechanism for bradykinin (BK) to stimulate intestinal secretomotor neurons and intestinal chloride secretion. Muscle-stripped guinea pig ileal preparations were mounted in Ussing flux chambers for the recording of short-circuit current (Isc). Basal Isc and Isc stimulated by BK when preincubated with the BK receptors antagonist and other chemicals were recorded using the Ussing chamber system. Prostaglandin E2 (PGE2) production in the intestine was determined by enzyme immunologic assay (EIA). Application of BK or B2 receptor (B2R) agonist significantly increased the baseline Isc compared to the control. B2R antagonist, tetrodotoxin and scopolamine (blockade of muscarinic receptors) significantly suppressed the increase in Isc evoked by BK. The BK-evoked Isc was suppressed by cyclooxygenase (COX)-1 or COX-2 specific inhibitor as well as nonselective COX inhibitors. Preincubation of submucosa/mucosa preparations with BK for 10 min significantly increased PGE2 production and this was abolished by the COX-1 and COX-2 inhibitors. The BK-evoked Isc was suppressed by nonselective EP receptors and EP4 receptor antagonists, but selective EP1 receptor antagonist did not have a significant effect on the BK-evoked Isc. Inhibitors of PLC, PKC, calmodulin or CaMKII failed to suppress BK-induced PGE2 production. The results suggest that BK stimulates neurogenic chloride secretion in the guinea pig ileum by activating B2R, through COX increasing PGE2 production. The post-receptor transduction cascade includes activation of PLC, PKC, CaMK, IP3 and MAPK.

An homogeneous population of beta 1-adrenoceptors subserves inhibitory responses in guinea-pig ileal preparations.

In transmurally stimulated guinea-pig ileal preparations, the pA2 values obtained with the selective beta 1- and beta 2-adrenoceptor antagonists atenolol and ICI 118,551 were independent of the beta-adrenoceptor agonist used [-)-isoprenaline or fenoterol) and were similar to values reported in isolated cardiac tissue. On the basis of these results, the relaxant responses to beta-adrenoceptor agonists in guinea-pig ileal preparations appear to be mediated by an homogeneous population of beta 1-adrenoceptors.

Investigation of the antimuscarinic and other actions of proadifen in-vitro.

The possibility that proadifen (SKF 525A) antagonizes endothelium-dependent relaxations to acetylcholine (ACh) in isolated blood vessel preparations via a muscarinic receptor blocking action has been investigated. In phenylephrine-contracted rat isolated aortic ring preparations (with endothelium), proadifen (10-100 microM) shifts ACh relaxant curves to the right without affecting the maximal response, yet endothelium-dependent relaxations to ATP are unaffected. At lower concentrations, proadifen (1-10 microM) antagonizes negative inotropic responses to ACh and ATP in guinea-pig left atria, antagonizes contractile responses to ACh and elevated [K+] in guinea-pig ileal preparations, displaces (-)-[3H]quinuclidinyl benzilate from muscarinic binding sites in membrane homogenates of guinea-pig ileal longitudinal muscle and reduces contractile responses to elevated [K+] in rat aortic ring preparations. It is concluded that proadifen may possess complex interactions with muscarinic receptors and Ca2+ entry blocking properties in concentrations 10-100 times lower than those reported to inhibit cytochrome P450-catalysed reactions.

Synthesis and pharmacology of 1-(2-thioxopyrrolidino)-4-pyrrolidino-2-butyne, the thiolactam analogue of oxotremorine.

Abstract The thiolactam analogue of oxotremorine (I) has been prepared and examined for muscarinic activity in mice and on guinea-pig ileal preparations. It is inactive in itself, but is transformed in vivo to oxotremorine (II), which has been identified in the urine of mice. The urine also contains 1-(2-oxopyrrolidino)-4-pyrrolidino-2-butanone (III) which is formed non-enzymatically from I under acidic conditions, but does not display any acetylcholine-like properties in vivo or in vitro. Oxotremorine is not formed from I in vitro.

Mucosal stimulation activates secretomotor neurons via long myenteric pathways in guinea pig ileum

This study examined whether mucosal stimulation activates long secretomotor neural reflexes and, if so, how they are organized. The submucosa of in vitro full thickness guinea pig ileal preparations was exposed in the distal portion and intracellular recordings were obtained from electrophysiologically identified secretomotor neurons. Axons in the intact mucosa of the oral segment were stimulated by a large bipolar stimulating electrode. In control preparations, a single stimulus pulse evoked a fast excitatory postsynaptic potential (EPSP) in 86% of neurons located 0.7-1.0 cm anal to the stimulus site. A stimulus train evoked multiple fast EPSPs, but slow EPSPs were not observed. To examine whether mucosal stimulation specifically activated mucosal sensory nerve terminals, the mucosa/submucosa was severed from the underlying layers and repositioned. In these preparations, fast EPSPs could not be elicited in 89% of cells. Superfusion with phorbol dibutyrate enhanced excitability of sensory neurons and pressure-pulse application of serotonin to the mucosa increased the fast EPSPs evoked by mucosal stimulation, providing further evidence that sensory neurons were involved. To determine whether these reflexes projected through the myenteric plexus, this plexus was surgically lesioned between the stimulus site and the impaled neuron. No fast EPSPs were recorded in these preparations following mucosal stimulation whereas lesioning the submucosal plexus had no effect. These results demonstrate that mucosal stimulation triggers a long myenteric pathway that activates submucosal secretomotor neurons. This pathway projects in parallel with motor and vasodilator reflexes, and this common pathway may enable coordination of intestinal secretion, blood flow, and motility.

Sensory peptide neurotransmitters mediating mucosal and distension evoked neural vasodilator reflexes in guinea pig ileum

The aim was to determine the role CGRP and/or tachykinins released from sensory neural mechanisms in enteric neural vasodilator pathways. These pathways project through the myenteric plexus to submucosal vasodilator neurons. Submucosal arterioles were exposed in the distal portion of an in vitro combined submucosal-myenteric guinea pig ileal preparation, and dilation was monitored with videomicroscopy. Vasodilator neural reflexes were activated by gently stroking the mucosa with a fine brush or by distending a balloon placed beneath the flat-sheet preparation in the proximal portion. Dilations evoked by mucosal stroking were inhibited 64% by the CGRP 8-37 and 37% by NK3 (SR 142801) antagonists. When the two antagonists were combined with hexamethonium, only a small vasodilation persisted. Balloon distension-evoked vasodilations were inhibited by NK3 antagonists (66%) but were not altered by CGRP 8-37. In preparations in which myenteric descending interneurons were directly activated by electrical stimulation, combined application of CGRP 8-37 and the NK antagonists had no effect. Stimulation of capsaicin sensitive nerves in the myenteric plexus did not activate these vasodilator reflexes. These findings suggest that mucosal-activated reflexes result from the release of CGRP and tachykinins from enteric sensory neurons. Distension-evoked responses were significantly blocked by NK3 antagonists, suggesting that stretch activation of myenteric sensory neurons release tachykinins that activate NK3 receptors on myenteric vasodilator pathways.

Properties of synaptic inputs from myenteric neurons innervating submucosal S neurons in guinea pig ileum.

This study examined synaptic inputs from myenteric neurons innervating submucosal neurons. Intracellular recordings were obtained from submucosal S neurons in guinea pig ileal preparations in vitro, and synaptic inputs were recorded in response to electrical stimulation of exposed myenteric plexus. Most S neurons received synaptic inputs [>80% fast (f) excitatory postsynaptic potentials (EPSP), >30% slow (s) EPSPs] from the myenteric plexus. Synaptic potentials were recorded significant distances aboral (fEPSPs, 25 mm; sEPSPs, 10 mm) but not oral to the stimulating site. When preparations were studied in a double-chamber bath that chemically isolated the stimulating "myenteric chamber" from the recording side "submucosal chamber," all fEPSPs were blocked by hexamethonium in the submucosal chamber, but not by a combination of nicotinic, purinergic, and 5-hydroxytryptamine-3 receptor antagonists in the myenteric chamber. In 15% of cells, a stimulus train elicited prolonged bursts of fEPSPs (>30 s duration) that were blocked by hexamethonium. These findings suggest that most submucosal S neurons receive synaptic inputs from predominantly anally projecting myenteric neurons. These inputs are poised to coordinate intestinal motility and secretion.

Involvement of Dihydropyridine‐Sensitive Ca2+ Channels in Adenosine‐Evoked Inhibition of Acetylcholine Release from Guinea Pig Ileal Preparation

The effects of adenosine and nifedipine on endogenous acetylcholine (ACh) release evoked by electrical stimulation from guinea pig ileal longitudinal muscle preparations exposed to physostigmine were evaluated using an HPLC with electrochemical detection (ECD) system. Resting ACh release, which was sensitive to tetrodotoxin (0.3 microM), was enhanced by Bay K 8644 (0.5 microM; a Ca2+ antagonist) or 4-aminopyridine (30 microM; a K+ channel blocker) but not by theophylline (100 microM; a P1 purinoceptor antagonist) or atropine (0.3 microM). The enhancement of the resting ACh release by Bay K 8644 was virtually unaffected by atropine. Electrically evoked ACh release was enhanced by around two- to fourfold in the presence of theophylline, atropine, Bay K 8644, 4-aminopyridine, or atropine. On the other hand, the evoked ACh release was reduced by adenosine (10-30 microM), nifedipine (0.1-0.3 microM; a dihydropyridine Ca2+ channel antagonist), or bethanechol (1-3 microM) in a concentration-related fashion. The reduction induced by adenosine or nifedipine was almost abolished by either theophylline or Bay K 8644, whereas that induced by bethanechol was virtually unaffected by these drugs. The inhibition by adenosine of ACh release was not influenced in the presence of 4-aminopyridine or atropine. However, this inhibition by adenosine was considerably enhanced by halving the Ca2+ concentration in the Krebs solution and was diminished by doubling the Ca2+ concentration. These findings suggest that adenosine produces a cholinergic neuromodulation presumably via modifying dihydropyridine-sensitive Ca2+ channel activities in the cholinergic neurons, and thus L-type Ca2+ channels may exist on the nerve terminals.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of newly synthesized N-cyclopropylmethyl derivatives of (−)-6β-acetylthionormorphine with opioid receptors

1. Analgesic activities of N-cyclopropylmethyl derivatives of (−)-6β-acetylthionormorphine, KT-89 and KT-90 and their interactions with opioid receptors were studied. 2. KT-89 and KT-90, as well as morphine inhibited the twitch response of the guinea-pig ileal preparation to electrical stimulation. Their pD 2 values indicated that KT-89 and KT-90 are about 6.5 and 10 times as potent as morphine, respectively. In guinea-pig ileal preparation KT-89 and KT-90 also behaved as a μ-antagonist. 3. In rabbit vas deferens which contains κ-receptors, these substances inhibited the twitch response to electrical stimulation and were about 6 times as potent as dynorphin. 4. Their effects on specific binding of [ 3H]naloxone (μ-selective ligand), [ 3H]ethylketocyclazocine (κ-selective ligand) and [ 3H] d-Ala 2- d-Leu 5-enkephalin (δ-selective ligand) to the synaptosomal fractions from rat brain were tested. Though both drugs had a nonselectively high affinity to μ-, κ- and δ-receptors, affinities of KT-89 and KT-90 to κ-receptors were about 6 and 13 times higher than that of morphine, respectively. 5. Analgesic activities of KT-89 and KT-90 were 6 and 10 times as potent as morphine in an acetic acid-induced writhing test, and 4 and 5 times as potent in a pressure test. 6. The present results suggest that KT-89 and KT-90 induced analgesic actions are mediated through an activation of κ-receptors. Both the drugs acted as δ-receptor antagonists. Further experiments are needed to study effects of their property as a δ-antagonist on analgesic action.

Pharmacological properties of newly synthesized derivatives of (−)-6β-acetylthionormorphine and their interactions with opioid receptors

1. Some pharmacological properties of newly synthesized derivatives of (−)-6β-acetylthionormorphine, AcS-morphine (KT-88), KT-89 and KT-90 and their interactions with opioid receptors were studied. 2. AcS-morphine was about twice as potent as morphine in the inhibitory action of the twitch response of the guinea-pig ileal preparation to electrical stimulation and about 5 times as potent as morphine in the analgesic action in the rats. Both the effects of AcS-morphine were inhibited by naloxone, suggesting that the site of action of AcS-morphine is μ-receptors. 3. Analgesic activities of KT-89 and KT-90 were about 6 and 10 times as potent as morphine in an acetic acid-induced writhing test and about 4 and 5 times as potent as in a pressure test. 4. As the analgesic action of KT-90 was antagonized by norbinaltorphimine, the site of action of KT-90 is concluded to be κ-receptors. Furthermore, KT-89 and KT-90 behaved as antagonists on μ-receptors. 5. The present results suggest that AcS-morphine which is N-methyl derivative, as well as morphine, has a selectively high affinity to μ-receptors, and that KT-89 and KT-90 which are N-cyclopropylmethyl derivatives have nonselectively high affinities to μ-, κ- and δ-receptors.

Organ‐bath studies using the irreversible β‐adrenoreceptor antagonist bromoacetylalprenololmenthane (BAAM)

1. The effects of the irreversible beta-adrenoreceptor antagonist bromoacetylalprenololmenthane (BAAM) were studied in isolated cardiac and uterine preparations from guinea-pigs and rats and in guinea-pig ileal preparations. 2. In the presence of BAAM (0.1-10 microM) concentration-effect curves to (-)-isoprenaline were shifted to the right in a concentration-dependent manner in all cardiac and uterine tissues. Maximum responses to (-)-isoprenaline were unaffected by BAAM except in guinea-pig left atrial and in some guinea-pig uterine preparations; however, the reductions in the maximum responses were not concentration-dependent. The mean pKB values for BAAM in guinea-pig left atria, right atria, rat whole atria and rat uterus were 7.26, 7.24, 6.84 and 7.90 respectively. 3. In guinea-pig ileal preparations, BAAM (0.1-30 microM) relaxed contractions induced by K+, histamine and acetylcholine in a non-beta-adrenoreceptor-related manner since relaxant responses were unaffected by propranolol (0.5 microM). In other tissues higher concentrations of BAAM (30-100 microM) elicited atrial standstill and depressed K+-induced contractions in uterine smooth muscle. 4. Treatment of tissues with BAAM (10-100 microM) followed by extensive wash-out increased the EC50 values for (-)-isoprenaline 21- to 83-fold. The maximum response to the catecholamine was unaffected by BAAM except in guinea-pig left atrial preparations following treatment with 100 microM BAAM. At these concentrations BAAM markedly increased the effective refractory period. 5. Concentration-effect curves for the partial agonist, oxymethylene-isoprenaline (OM-ISO) were shifted to the right 12- to 355-fold after pretreatment of tissues with BAAM (10-30 microM) followed by wash-out. The maximum response to OM-ISO was unaltered in guinea-pig and rat uteri and was reduced to a similar degree as observed with (-)-isoprenaline in guinea-pig left atria. 6. In general, the non-selective beta-adrenoreceptor antagonist BAAM depressed maximum responses to beta-adrenoreceptor agonists only in cardiac preparations and at concentrations which elicited depressant activity. On the basis of the present study, BAAM does not appear to be a suitable irreversible beta-adrenoreceptor antagonist for use in organ bath experiments.

Effects of phenoxybenzamine on responses to some receptor agonists and calcium in vitro.

Noradrenaline-induced contractions of the rabbit and rat isolated aorta and guinea-pig spleen strips were inhibited by concentrations of phenoxybenzamine which did not affect responses to calcium. This may suggest a specific action on alpha-adrenoceptors. However, analysis of noradrenaline concentration-effect curves in guinea-pig spleen indicated that 1 mumol/l phenoxybenzamine should have reduced the available receptor population to 6% of control, but data from radioligand binding experiments on the same tissues using [3H]-prazosin indicated a reduction of the receptor population to only 82% of control. The reduced responsiveness observed in the organ bath study after phenoxybenzamine pretreatment, whilst not apparently related to effects on voltage-dependent calcium channels, could be due to the actions of phenoxybenzamine on other (non-receptor) processes such as receptor-operated calcium channels. Maximal contractile responses to histamine in rabbit isolated aorta but not those in guinea-pig isolated ileal preparations, were depressed by concentrations of phenoxybenzamine which depressed responses to calcium. Phenoxybenzamine produced parallel rightward shifts of curves to carbachol in guinea-pig ileal preparations but only depressed maximal responses to the agonist in higher concentrations which reduced responses to calcium. On the basis of the results obtained with calcium it is possible that the effects of phenoxybenzamine on receptor-mediated responses could be produced through the actions of this antagonist at less specific sites such as voltage-dependent calcium channels for histamine in rabbit aorta and carbachol in guinea-pig ileum. For alpha-receptor mediated responses in aortic and splenic strip preparations and for histamine-mediated responses in guinea-pig ileum, the actions of phenoxybenzamine may reflect an interaction of the antagonist with receptor-operated calcium channels.

Pre- and postjunctional effects of clonidine- and oxymetazoline-like compounds in guinea-pig ileal preparations.

1 Noradrenaline and 28 imidazolidine (clonidine-like) and imidazoline (oxymetazoline-like) compounds with various phenyl ring substituents have been examined for their ability to inhibit responses to transmural stimulation and exogenous acetylcholine in ileal preparations from reserpine-treated guinea-pigs.2 The bathing solution contained propranolol, mepyramine, cimetidine and desipramine to preclude interference with the responses by other than the alpha-receptor-mediated actions of the compounds.3 In transmurally stimulated preparations the inhibitory response to noradrenaline is due to a combination of prejunctional alpha-adrenoceptor stimulation and a postjunctional depressant effect that does not involve adrenoceptor activation.4 Of the 28 imidazolidines and imidazolines studied, 21 inhibited transmurally elicited responses. In the various compounds studied this effect involved actions at pre- or postjunctional sites as indicated by (a) the frequency-dependence of the inhibitory response, (b) its susceptibility to blockade by alpha-receptor antagonists and (c) the relative concentrations required to inhibit responses to transmural stimulation and exogenous acetylcholine.

Reversible depression of electrically-induced contractions of guinea pig longitudinal muscle by a reversible inhibitor of prostaglandin synthetase

The pesticide chlordimeform (CUM) depressed the electrically-induced twitch responses of the guinea pig longitudinal muscle ED 50 = 3 × 10 −5 M. Naloxone reversed twitch depression induced by morphine but not by CDM. Phentolamine reversed twitch depression induced by either norepinephrine or clonidine, but not by CDM. PGE2 completely reversed twitch depression induced by either CDM or indomethacin, but only partially reversed twitch depression induced by lidocaine. The actions of CDM best resemble those of the nonsteroidal anti-inflammatory agents. The reversibility of the CDM depression by washing indicates that the guinea pig ileal preparation is a convenient screen for distinguishing reversible from irreversible inhibitors of prostaglandin biosynthesis.