Muscle Strips Of Guinea-pig Ileum Research Articles

Possible role of prostaglandins in post-tetanic potentiation at the nerve-muscle junction in the longitudinal muscle strip of guinea-pig ileum.

The effect of tetanic stimulation on the twitch responses of the longitudinal muscle-myenteric plexus preparation of the guinea-pig ileum to electrical stimulation was investigated in the presence of naloxone. Under this condition, or after the addition of PGE2, twitch contractions were maximal and no potentiation of twitches following tetanus was observed. In the presence of indomethacin (1 mumol litre-1) twitches were diminished and post-tetanic potentiation (PTP) was manifested. PTP was seen with indomethacin concentrations of 1 to 20 mumol litre-1 or after simultaneous addition of diphloretin phosphate (16 mumol litre-1). Thus it seems unlikely that the effect of prostaglandins released during tetanic stimulation would be of key importance for the manifestation of PTP. Rather it is thought that a decrease in the release of acetylcholine from motor nerve terminals, and consequently smaller twitches in the presence of indomethacin, offer favourable conditions for PTP.

Reply to Dodds & Rivory

British Journal of Pharmacology (2001) 134, 449–450; doi:10.1038/sj.bjp.0704283

Receptor constants for endomorphin-1 and endomorphin-1-ol indicate differences in efficacy and receptor occupancy

The opioid properties of endomorphin derivatives containing a C-terminal alcoholic(-ol) function were compared to the parent amidated compounds in isolated organs (longitudinal muscle strip of guinea-pig ileum and mouse vas deferens). Similar data were also generated for the μ-opioid receptor selective agonist synthetic peptide ( d-Ala 2, MePhe 4, Gly 5-ol)-enkephalin (DAMGO) and its Gly 5-NH 2 congener (DAMGA). Endomorphin-1-ol (Tyr-Pro-Trp-Phe-ol) had an IC 50 of 80.6 nM in mouse vas deferens and 61.2 nM in guinea-pig ileum; the corresponding values for endomorphin-2-ol (Tyr-Pro-Phe-Phe-ol) were 49.6 and 48.2 nM, for DAMGO 59.8 and 29.2 nM, respectively. As it was indicated by the antagonism by naltrexone, the agonist actions were exerted exclusively at μ-opioid receptors in both organs. The -ol derivatives were slightly (2.3–4.3 times) less potent than the parent amides in the bioassays: all peptides had, apparently, full agonist properties in intact preparations. With the aim of revealing potential partial agonist properties among the investigated peptides, we partially inactivated the μ-opioid receptor pool in mouse vas deferens by 5×10 −7 M β-funaltrexamine. The calculated receptor constants indicated a “high-affinity, low intrinsic efficacy” profile (i.e. a potential partial agonist property) for endomorphin-1, an intermediate character for endomorpin-1-ol and full agonism for DAMGA and DAMGO. Apparently, a higher receptor fraction remained accessible for endomorphin-1 (42.8%) than for the -ol congener (14.0%), DAMGO (20.2%) and DAMGA (14.1%) after partial inactivation.

Flurorescent derivatives of bovine neurotensin 8–13 fragment

Fluorescent derivatives of bovine neurotensin 8–13 fragment were prepared. For N-terminal labelling, 4-[7-hydroxycoumaryl]acetic acid (Hca), 4-[7-methoxycoumaryl]acetic acid (Mca) and 2-amino-3-[4-[7-methoxycoumaryl]]propionic acid (Amp) were used while the C-terminus of the peptide chain was elongated with Amp. The fluorescence excitation and emission spectra of the peptide derivatives were studied. Hca- and Mca/Amp-derivatives were easily distinguishable because of the 60 nm shift of their emission maxima. Compared with the natural sequence, the presence of an N-terminal label did not influence the biological potency in a longitudinal muscle strip of guinea-pig ileum, while labelling at the C-terminus considerably reduced the activity of the peptide.

Fluorescent derivatives of bovine neurotensin 8–13 fragment

Fluorescent derivatives of bovine neurotensin 8–13 fragment were prepared. For N-terminal labelling, 4-[7-hydroxycoumaryl]acetic acid (Hca), 4-[7-methoxycoumaryl]acetic acid (Mca) and 2-amino-3-[4-[7-methoxycoumaryl]]propionic acid (Amp) were used while the C-terminus of the peptide chain was elongated with Amp. The fluorescence excitation and emission spectra of the peptide derivatives were studied. Hca- and Mca/Amp-derivatives were easily distinguishable because of the 60 nm shift of their emission maxima. Compared with the natural sequence, the presence of an N-terminal label did not influence the biological potency in a longitudinal muscle strip of guinea-pig ileum, while labelling at the C-terminus considerably reduced the activity of the peptide.

A1-receptor-mediated effect of adenosine on the release of acetylcholine from the myenteric plexus: role and localization of ecto-ATPase and 5'-nucleotidase.

No attempt has been made so far to classify the subtypes of presynaptic inhibitory adenosine receptors located in the myenteric plexus and to localize ecto-ATPase and 5'-nucleotidase in the intestine. The release of [3H]acetylcholine and smooth muscle responses to acetylcholine were measured and the effect of selective adenosine receptor ligands was studied using field-stimulated isolated longitudinal muscle strips of guinea-pig ileum. Release of ATP and its hydrolysis rate were also measured using the luciferin-luciferase technique. A histochemical method combined with electron microscopy was used for localization of ecto-ATPase and 5'-nucleotidase, enzymes responsible for destruction of extracellular ATP, ADP and AMP. Subtype-selective A1-receptor agonists and antagonists inhibited and enhanced, respectively, the release of acetylcholine associated with neuronal activity. A significant amount of ATP was released in response to electrical stimulation and administration of carbamylcholine. The release of ATP was inhibited by atropine and 4-diphenylacetoxy-N-methylpiperidine methiodide, an M3-receptor antagonist. Hydrolysis of ATP was rapid and resulted in an accumulation of extracellular adenosine involved in presynaptic A1-receptor-mediated inhibition of acetylcholine release. While the inhibitory effect of adenosine and ATP was significantly potentiated by dipyridamol, an adenosine uptake blocker, that of 2-ms ATP was not. The effect of ATP was not competitively antagonized by 8-cyclopentyl-1,3-dipropylxanthine, a selective A1-receptor antagonist. In conclusion, axon terminals of cholinergic interneurons are equipped with inhibitory A1- and P2 gamma-receptors. Therefore, both adenosine and ATP control the release of acetylcholine through these receptors. ATP is mainly released from the smooth muscle in response to stimulation of M3-muscarinic receptors by endogenous acetylcholine (cascade transmission [Vizi E. S. et al. (1992) Neuroscience 50, 455-465]) and is rapidly hydrolysed by ecto-ATPase localized on the surface of the smooth muscle and axon terminals producing ADP and AMP, and by 5'-nucleotidase present only on the surface of smooth muscle cells producing adenosine.

Functional and neurochemical evidence that neurotensin-induced release of acetylcholine from Auerbach's plexus of guinea-pig ileum is presynaptically controlled via ?2-adrenoceptors

The effect of neurotensin (NT) on [3H]acetylcholine release and contraction from isolated longitudinal muscle strip of guinea-pig ileum was examined. Neurotensin dose-dependently enhanced the release of [3H]-acetylcholine. This effect of neurotensin was inhibited by stimulation of alpha 2-adrenoceptors: noradrenaline, clonidine, xylazine or dexmedetomidine (alpha 2-adrenoceptor agonists) inhibited neurotensin-induced release of acetycholine (ACh) as well as the contractions, while CH-38083 or yohimbine (alpha 2-adrenoceptor antagonist) prevented this inhibitory effect. Our findings suggest that neurotensin may play a neuromodulatory role in the regulation of cholinergic neuronal activity in the gut and this modulatory effect is continuously controlled by the tonic activity of the sympathetic nervous system: endogenous noradrenaline release is capable of reducing the release of ACh and the consequent contraction of the gut enhanced by neurotensin.

Functional evidence that acetylcholine release from Auerbach's plexus of guinea-pig ileum is modulated by α 2A-adrenoceptor subtype

The effects of α 2-adrenoceptor ligands on electrically stimulated [ 3H]acetylcholine release from longitudinal muscle strips of guinea-pig ileum were examined. Xylazine or oxymetazoline reduced the release of [ 3H]acetylcholine inn a concentration-dependent manner, both these actions being antagonized by idazoxan, CH 38083, or WB 4101. Prazosin. considered as an α 2B-adrenoceptor antagonist, failed to modify the inhibitory effects of xylazine or oxymetazoline. It is concluded that the α 2-adrenoceptors involved in the modulation of acetylcholine release from cholinergic axon terminals of guinea-pig ileum are of the α 2A subtype.

Pharmacological differentiation of presynaptic M 1 muscarinic receptors modulating acetylcholine release from postsynaptic muscarinic receptors in guinea-pig ileum

1. 1. Effects of three muscarinic antagonists on electrically evoked ACh release and contractile response were investigated in longitudinal muscle strips of guinea-pig ileum suspended in an organ-bath and superfused with Krebs solution. ACh release was determined by a specific radioimmunoassay. 2. 2. Telenzepine, a selective M 1 muscarinic antagonist, increased the ACh release at a concentration of 100-fold less than that inhibiting the contractile response (10 vs 1000 nM). 3. 3. AF-DX 116, a cardioselective M 2 muscarinic antagonist, inhibited the contractile response at 10 μM, but did not affect the ACh release at this concentration. 4. 4. (−) N-Methylscopolamine (NMS) did not affect the ACh release, but inhibited the contractile response at all concentrations tested (1–1000 nM), indicating (−)NMS can be used as an ileal specific postsynaptic muscarinic antagonist. 5. 5. These data demonstrate that presynaptic muscarinic receptors modulating ACh release are distinct from postsynaptic ones involved in the contractile response and can be classified as M 1 subtype.

Antinociception induced by systemic administration of local anaesthetics depends on a central cholinergic mechanism.

1 The antinociceptive effects of systemically-administered procaine, lignocaine and bupivacaine were examined in mice and rats by using the hot-plate, writhing and tail flick tests. 2 In both species all three local anaesthetics produced significant antinociception which was prevented by atropine (5 mg kg-1, i.p.) and by hemicholinium-3 (1 microgram per mouse, i.c.v.), but not by naloxone (3 mg kg-1, i.p.), alpha-methyl-p-tyrosine (100 mg kg-1, s.c.), reserpine (2 mg kg-1, i.p.) or atropine methylbromide (5.5 mg kg-1, i.p.). 3 Atropine (5 mg kg-1, i.p.) which totally antagonized oxotremorine (40 micrograms kg-1, s.c.) antinociception did not modify morphine (5 mg kg-1, s.c.) or baclofen (4 mg kg-1, s.c.) antinociception. On the other hand, hemicholinium, which antagonized local anaesthetic antinociception, did not prevent oxotremorine, morphine or baclofen antinociception. 4 Intracerebroventricular injection in mice of procaine (200 micrograms), lignocaine (150 microgram) and bupivacaine (25 micrograms), doses which were largely ineffective by parenteral routes, induced an antinociception whose intensity equalled that obtainable subcutaneously. Moreover, the i.c.v. injection of antinociceptive doses did not impair performance on the rota-rod test. 5 Concentrations below 10(-10) M of procaine, lignocaine and bupivacaine did not evoke any response on the isolated longitudinal muscle strip of guinea-pig ileum, or modify acetylcholine (ACh)-induced contractions. On the other hand, they always increased electrically-evoked twitches. 6 The same concentrations of local anaesthetics which induced antinociception did not inhibit acetylcholinesterase (AChE) in vitro. 7 On the basis of the above findings and the existing literature, a facilitation of cholinergic transmission by the local anaesthetics is postulated; this could be due to blockade of presynaptic muscarinic receptors.

Inhibition by p-nitrophenylphosphate of acetylcholine release induced by Na +-deprivation

The effect of p-nitrophenylphosphate ( p-NPP) on the release of acetylcholine evoked by drugs and ionic environments known to inhibit Na +, K +-ATPase was studied in isolated cortical slices of rat brain and longitudinal muscle strip of guinea-pig ileum. p-NPP inhibited the release of acetylcholine induced by sodium deprivation provided that the circumstances were in favour of the function of the K +-activated part of ATPase. However, it failed to antagonize the increase in the acetylcholine release elicited by omission of K + or by administration of ouabain. Therefore it is concluded that the K +-stimulated phosphatase moiety of the Na +, K +-ATPase might be involved in the release of acetylcholine.

1,4-Dithiothreitol-induced changes in histamine H1-agonist efficacy and affinity in the longitudinal smooth muscle of guinea-pig ileum.

The effect of 1,4-dithiothreitol (DTT) on histamine H1-receptor agonist affinity and efficacy has been investigated in longitudinal muscle strips of guinea-pig ileum. Exposure of ileal smooth muscle to DTT significantly increased the maximal responses to the partial agonists SKF71473 and DE-2PEA, indicative of an increase in agonist efficacy. This effect was paralleled by a small decrease in EC50 values. In contrast, DTT produced a parallel displacement of the concentration-response curve to the full agonist histamine in the same muscle strips. Studies in which phenoxybenzamine and benzilylcholine mustard were used to reduce the maximum response to histamine suggested that DTT altered both agonist affinity and efficacy. The affinity constant for histamine, calculated by the method of Furchgott & Bursztyn (1967), increased by 2.7 fold in the presence of DTT. Furthermore, agonist efficacy also appeared to increase in the presence of DTT since the maximum response to histamine following phenoxybenzamine treatment increased on application of DTT. [3H]-mepyramine binding studies confirmed that DTT increased agonist affinity. DTT produced a significant parallel shift to the left of the displacement curves for histamine, 2-methylhistamine, 2-pyridylethylamine and 2-thiazolylethylamine. The results of this study therefore suggest that DTT potentiates H1-receptor-mediated contractile activity in guinea-pig ileum by increasing both agonist efficacy and affinity.

O-307 - Enhancement of acetylcholine release and contraction response by pirenzepine and atropine in the longitudinal muscle strips of guinea-pig ileum

O-307 - Enhancement of acetylcholine release and contraction response by pirenzepine and atropine in the longitudinal muscle strips of guinea-pig ileum

Presynaptic effects of neuropeptide Y on [ 3H]noradrenaline and [ 3H]acetylcholine release

The electrically evoked release of radioactivity from mouse vas deferens and rat hypothalamic slices preloaded with [ 3H]noradrenaline was measured. In addition the release of [ 3H]acetylcholine from longitudinal muscle strip of guinea-pig ileum was also measured. Neurochemical evidence has been obtained that neuropeptide Y (NPY), although it co-exists and is released with (−)-noradrenaline (NA), it behaves differently as far as its effects on presynaptic modulation of chemical neurotransmission is concerned. It exerts a frequency-dependent presynaptic inhibitory effect on noradrenaline release from mouse vas deferens but has no effect on the electrically evoked release of NA from rat hypothalamus. Unlike NA, NPY does not influence the release of [ 3H]acetylcholine from the longitudinal muscle strip of guinea-pig ileum and does not potentiate the presynaptic effect of NA. It seems very likely, that the inhibitory effect of NPY is mediated via receptors. Its action is concentration dependent. While exogenous noradrenaline inhibited the release of noradrenaline by 91%, the maximum inhibition reached with NPY was not higher than 60%, indicating that either the intrinsic activity of NPY is lower or much less axon terminals are equipped with NPY receptors. Peptide YY (PYY) also reduced the release of NA from mouse vas deferens.

Effects of Peptidase Inhibitors on the (Met5)-Enkephalin Hydrolysis in Ileal and Striatal Preparations of Guinea-Pig: Almost Complete Protection of Degradation by the Combination of Amastatin, Captopril and Thiorphan

AbstractThe contents of [Met5]-enkephalin and its four hydrolysis products. Tyr, Tyr-Gly, Tyr-Gly-Gly, and Tyr-Gly-Gly-Phe, in the sample obtained after enkephalin was incubated with tissues in either the absence or the presence of the peptidase inhibitor were estimated by high performance liquid chromatography combined with electrochemical detection in order to elucidate the effect of the peptidase inhibitor on enkephalin hydrolysis. Free Tyr was the major degradative product in the absence of the peptidase inhibitor, while the major hydrolysis product was the Tyr-Gly-Gly fragment in the presence of amastatin in both total homogenates and membrane fractions prepared from either the myenteric plexus-longitudinal muscle strip of guinea-pig ileum or the striatum of guinea-pig brain. Additionally. Captopril significantly decreased the generation of Tyr-Gly-Gly in the presence of amastatin in both ileal and striatal membrane fractions. Moreover, thiorphan significantly prevented the formation of Tyr-Gly-Gly in the presence of both amastatin and Captopril in both membrane preparations. Finally, when [Met6]-enkephalin was incubated with either an ileal or a striatal membrane fraction for 60 min at 37°C in the presence of three peptidase inhibitors, amastatin. Captopril, and thiorphan. approximately 98 or 94%, respectively, of enkephalin remained intact. The results indicated that [Met6]-enkephalin was almost exclusively hydrolyzed by three distinct enzymes, amastatin-sensitive ammopeplidase. Captopril-sensitive peptidyl dipeptidase A. and thiorphan-sensitive endopeptidase-24.11 in both ileal and striatal membrane fractions.

Is there functional cholinergic innervation in the frog duodenum ( Rana catesbiana)?

1. To determine if functional cholinergic innervation occurs in the frog duodenum or not, the effects of exogenous acetylcholine and electrical transmural stimulation, the contractile activity of an acid extract from the frog duodenum, and the distribution of acetylcholinesterase (AChE) activity in the wall of the frog duodenum were investigated. 2. Acetylcholine caused non-sustained contraction in a dose-dependent manner (100nM−1 mM). The ed 50) value was 17 ± 2.4 μM. Atropine (500 nM) shifted the dose-response curve for acetylcholine parallel to the right. 3. Transmural stimulation of the frog duodenum caused frequency-dependent (0.5–50 Hz) contraction which was not decreased by atropine (500 nM) at all. 4. The acid extract from the frog duodenum caused contraction of a longitudinal muscle strip of guinea-pig ileum but atropine (500 nM) had no significant effect on the contraction. 5. Only a little AChE activity was found in Auerbach's plexus of the frog duodenum compared with that of the rat ileum. 6. These results suggest that a cholinergic nerve is present in the frog duodenum but its physiological significance is very small.

Neurochemical evidence for the release of acetylcholine from the guinea-pig ileum myenteric plexus by capsaicin

Superfision of the sensory stimulant drug capsaicin (3.3 μM) released [ 3H]acetylcholine from isolated myenteric plexus-longitudinal muscle strips of guinea-pig ileum, preincubated with [ 3H]choline. This effect was prevented by tetrodotoxin. There was a fast tachyphylaxis to the effect of capsaicin. It is suggested that capsaicin-sensitive nerve endings capable of activating intrinsic cholinergic neurones are situated in the myenteric plexus of the guinea-pig ileum.

Met5-enkephalin-Arg6-Phe7 inhibition of noradrenaline and acetylcholine release from peripheral organs.

The effect of the opioid heptapeptide Met5-enkephalin-Arg6-Phe7 was measured on the contractions of the longitudinal muscle of the guinea-pig ileum and mouse vas deferens; on release of endogenous and newly synthesized acetylcholine from the longitudinal muscle strip preparation of the guinea-pig ileum and of 3H-noradrenaline from mouse vas deferens. Met5-enkephelin-Arg6-Phe7 depressed the contractions of the longitudinal muscle strip of guinea-pig ileum elicited by low frequency stimulation (IC50 366.5 +/- 71.5 nM). The Ke value of naltrexone against the heptapeptide was found to be 0.50 +/- 0.08 nM. Bestatin prolonged and strongly potentiated the action of the opioid peptide in this tissue while captopril proved to be only slightly effective. Met5-enkephalin-Arg6-Phe7 also inhibited neuroeffector transmission in the mouse vas deferens with an IC50 value of 9.44 +/- 2.24 nM. The Ke value of naltrexone was 6.33 +/- 1.22 nM against the heptapeptide. Met5-enkephalin-Arg6-Phe7 (10(-5) M) failed to affect the low frequency stimulated release of endogenous acetylcholine from the guinea-pig ileum preparation. Addition of either bestatin or captopril with the heptapeptide resulted in a reduction of acetylcholine output. The opioid peptide by itself decreased the release of newly synthesized acetylcholine outflow from this organ. Met5-enkephalin-Arg6-Phe7 also reduced the 3H-noradrenaline outflow from mouse vas deferens and this effect was antagonized by naltrexone. From our data we conclude that Met5-enkephalin-Arg6-Phe7 affects the neural transmission of peripheral autonomically innervated organs by depressing the release of neurotransmitter. In vitro it has enkephalin-like character with a preference for the so called delta receptor interaction.

Enkephalin-like character and analgesia

The opioid activities of enkephalin analogues bearing D- or L-aminopentane-sulfonic/phosphonic acid at position 5 were studied in vitro, in electrically stimulated longitudinal muscle strip of guinea-pig ileum and mouse vas deferens preparations and in vivo in the rat tail-flick test. Using their in vitro effects Met-enkephalin-like, β-endorphin-like, (nor)morphine-like and derivatives of intermediate character could be differentiated. Correlating the in vitro activities with the analgesic activity in vivo it is concluded that the enkephalin-like character in a pentapetide may hinder the expression of analgesic activity, when the compounds are given into the cerebroventicular system.

Correlation between circular dichroism data and biological activities of 2, 5 substituted enkephalin analogues

The relative structural rigidity of enkephalin analogues characterized by the molar ellipticity data obtained from the circular dichroism spectra of peptides was correlated with the opioid agonist activities of compounds displayed in isolated, electrically stimulated longitudinal muscle strip of guinea-pig ileum and mouse vas deferens preparations. It was found that the so called μ receptors modelled by guinea-pig ileum preferred the analogues with high capacity to exist in folded form, whilst the so called δ receptors (mouse vas deferens) accepted flexible ligands as readily as rigid ones.