1 Experiments were carried out in human detrusor strips to characterize muscarinic receptor subty... more 1 Experiments were carried out in human detrusor strips to characterize muscarinic receptor subtypes involved in the prejunctional regulation of acetylcholine (ACh) release from cholinergic nerve terminals, and in the postjunctional smooth muscle contractile response. 2 In detrusor strips preincubated with [ 3 H]-choline, electrical ®eld stimulation (600 pulses) delivered in six trains at 10 Hz produced a tritium out¯ow and a contractile response. In the presence of 10 mM paraoxon (to prevent ACh degradation) the tritium out¯ow was characterized by HPLC analysis as [ 3 H]-ACh (76%) and [ 3 H]-choline (24%).
The effects of norlloxacin and enoxacin were examined on spontaneous motor activity in the guinea... more The effects of norlloxacin and enoxacin were examined on spontaneous motor activity in the guinea-pig isolated ileum. Micromolar concentrations of both compounds caused a biphasic response consisting of relaxation followed by transient contraction. Relaxation to noriloxacin, which was unaffected by phentolamine, propranolol and hyoscine (each at 1 pM), was partially sensitive to tetrodotoxin (1 pM). This indicates that the response is partly mediated by nonadrenergic non-cholinergic (NANC) inhibitory nerves, and partly related to a direct action on the smooth muscle. Apamin (0.1 pM) and suramin (300 pM) inhibited norfloxacin-induced relaxations to an extent similar to that of tetrodotoxin. Conversely, NG-nitro-L-arginine (300 pM) was ineffective. In the presence of theophylline (100 pM) and 3-isobutyl-lmethylxanthine (1 0 pM), norfloxacin caused relaxation less effective than when added alone. Based on this observation, the NANC component of the relaxation apparently depends on ATP release, whereas the direct component might be due, at least in part, to phosphodiesterase inhibition. Norfloxacin-induced contractions were neurogenic and cholinergic in nature. They were reduced by indomethacin or S-ketoprofen (both at 0.01-1 pM) and suramin (300 pM), suggesting involvement of local prostaglandin production probably induced by ATP release. Previous findings revealed that norfloxacin acted as a non-competitive antagonist at enteric GABAA receptors. In this study the same property was shared by enoxacin against the contractile response to 3-aminopropane sulphonic acid (3-APS), a GABAA receptor agonist. In conclusion, fluoroquinolones exert inhibitory and excitatory effects in the guinea-pig ileum. These are mediated by ATE prostaglandin and acetylcholine release that might underlie, at least in part, the alterations of gastrointestinal motility observed after fluoroquinolone administration. Furthermore, isolated intestinal preparations might be useful to predict the GABAA-antagonist potential of this class of compounds.
Ischemic pain-examples include the chest pain of a heart attack and the leg pain of a 30 s sprint... more Ischemic pain-examples include the chest pain of a heart attack and the leg pain of a 30 s sprint-occurs when muscle gets too little oxygen for its metabolic need. Lactic acid cannot act alone to trigger ischemic pain because the pH change is so small. Here, we show that another compound released from ischemic muscle, adenosine tri-phosphate (ATP), works together with acid by increasing the pH sensitivity of acid-sensing ion channel number 3 (ASIC3), the molecule used by sensory neurons to detect lactic acidosis. Our data argue that ATP acts by binding to P2X receptors that form a molecular complex with ASICs; the receptor on sensory neurons appears to be P2X5, an electrically quiet ion channel. Coincident detection of acid and ATP should confer sensory selectivity for ischemia over other conditions of acidosis.
Abstract: The effects of norfloxacin and enoxacin were examined on spontaneous motor activity in ... more Abstract: The effects of norfloxacin and enoxacin were examined on spontaneous motor activity in the guinea-pig isolated ileum. Micromolar concentrations of both compounds caused a biphasic response consisting of relaxation followed by transient contraction. Relaxation to norfloxacin, which was unaffected by phentolamine, propranolol and hyoscine (each at 1 μM), was partially sensitive to tetrodotoxin (1 μM). This indicates that the response is partly mediated by non-adrenergic non-cholinergic (NANC) inhibitory nerves, and partly related to a direct action on the smooth muscle. Apamin (0.1 μM) and suramin (300 μM) inhibited norfloxacin-induced relaxations to an extent similar to that of tetrodotoxin. Conversely, NG-nitro-L-arginine (300 μM) was ineffective. In the presence of theophylline (100 μM) and 3-isobutyl-1-methylxanthine (10 μM), norfloxacin caused relaxation less effective than when added alone. Based on this observation, the NANC component of the relaxation apparently depends on ATP release, whereas the direct component might be due, at least in part, to phosphodiesterase inhibition. Norfloxacin-induced contractions were neurogenic and cholinergic in nature. They were reduced by indomethacin or S-ketoprofen (both at 0.01–1 μM) and suramin (300 μM), suggesting involvement of local prostaglandin production probably induced by ATP release. Previous findings revealed that norfloxacin acted as a non-competitive antagonist at enteric GABAA receptors. In this study the same property was shared by enoxacin against the contractile response to 3-aminopropane sulphonic acid (3-APS), a GABAA receptor agonist. In conclusion, fluoroquinolones exert inhibitory and excitatory effects in the guinea-pig ileum. These are mediated by ATP, prostaglandin and acetylcholine release that might underlie, at least in part, the alterations of gastrointestinal motility observed after fluoroquinolone administration. Furthermore, isolated intestinal preparations might be useful to predict the GABAA-antagonist potential of this class of compounds.
The effect of the melatonin receptor ligand, 2-phenylmelatonin, has been assessed in isolated str... more The effect of the melatonin receptor ligand, 2-phenylmelatonin, has been assessed in isolated strips of the guinea-pig proximal colon. 2-Phenylmelatonin (0.01 nM-1 μM) caused a concentration-dependent contractile response. The potency value (−log EC50) was 9.3±1.0. The maximum effect was 25±4% of that elicited by the maximally effective concentration (0.3 μM) of 5-HT and 43±3% of that by the maximally effective concentration (10 μM) of melatonin. When used as an antagonist, 2-phenylmelatonin (0.01 nM and 0.1 nM) concentration-dependently inhibited melatonin-induced contractions with depression of the maximum response by 25% and 54%, respectively. Higher (1 nM) 2-phenylmelatonin concentrations failed to antagonize melatonin-induced response. Prazosin (0.3 μM), a selective antagonist of melatonin MT3 sites, antagonized melatonin-induced contractions to an extent similar to that induced by 0.01 nM 2-phenylmelatonin (with 30% reduction of the maximum effect to melatonin). The combination of 0.3 μM prazosin and 0.01 nM 2-phenylmelatonin caused antagonism similar in extent to that caused by each individual antagonist. 2-Phenylmelatonin at subnanomolar concentrations behaves as an antagonist of melatonin-induced contractile responses while at nanomolar/micromolar concentrations it behaves as a weak contractile agonist.
The effect of the melatonin receptor ligand, 2-phenylmelatonin, has been assessed in isolated str... more The effect of the melatonin receptor ligand, 2-phenylmelatonin, has been assessed in isolated strips of the guineapig proximal colon. 2-Phenylmelatonin (0.01 nM-1 mM) caused a concentration-dependent contractile response. The potency value (ªlog EC 50 ) was 9.3∫1.0. The maximum effect was 25∫4% of that elicited by the maximally effective concentration (0.3 mM) of 5-HT and 43∫3% of that by the maximally effective concentration (10 mM) of melatonin. When used as an antagonist, 2-phenylmelatonin (0.01 nM and 0.1 nM) concentration-dependently inhibited melatonin-induced contractions with depression of the maximum response by 25% and 54%, respectively. Higher (1 nM) 2-phenylmelatonin concentrations failed to antagonize melatonin-induced response. Prazosin (0.3 mM), a selective antagonist of melatonin MT 3 sites, antagonized melatonin-induced contractions to an extent similar to that induced by 0.01 nM 2-phenylmelatonin (with 30% reduction of the maximum effect to melatonin). The combination of 0.3 mM prazosin and 0.01 nM 2-phenylmelatonin caused antagonism similar in extent to that caused by each individual antagonist. 2-Phenylmelatonin at subnanomolar concentrations behaves as an antagonist of melatonin-induced contractile responses while at nanomolar/micromolar concentrations it behaves as a weak contractile agonist.
The morphological pattern and motor correlates of nitric oxide (NO) and vasoactive intestinal pol... more The morphological pattern and motor correlates of nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) innervation in the human isolated gastric fundus was explored.By using the nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-diaphorase and specific rabbit polyclonal NO-synthase (NOS) and VIP antisera, NOS- and VIP-containing varicose nerve fibres were identified throughout the muscle layer or wrapping ganglion cell bodies of the myenteric plexus. NOS-immunoreactive (IR) neural cell bodies were more abundant than those positive for VIP-IR. The majority of myenteric neurones containing VIP coexpressed NADPH-diaphorase.Electrical stimulation of fundus strips caused frequency-dependent NANC relaxations. NG-nitro-L-arginine (L-NOARG: 300 μM) enhanced the basal tone, abolished relaxations to 0.3–3 Hz (5 s) and those to 1 Hz (5 min), markedly reduced (∼50%) those elicited by 10–50 Hz, and unmasked or potentiated excitatory cholinergic responses at frequencies 1 Hz. L-NOARG-resistant relaxations were virtually abolished by VIP (100 nM) desensitization at all frequencies.Relaxations to graded low mechanical distension (1 g) were insensitive to tetrodotoxin (TTX: 1 μM) and L-NOARG (300 μM), while those to higher distensions (2 g) were slightly inhibited by both agents to the same extent (∼25%).In the human gastric fundus, NOS- and VIP immunoreactivities are colocalized in the majority of myenteric neurones. NO and VIP mediate electrically evoked relaxations: low frequency stimulation, irrespective of the duration, caused NO release only, whereas shortlasting stimulation at high frequencies induced NO and VIP release. Relaxations to graded mechanical distension were mostly due to passive viscoelastic properties, with a slight NO-mediated neurogenic component at 2 g distension. The difference between NO and VIP release suggests that in human fundus accommodation is initiated by NO.The morphological pattern and motor correlates of nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) innervation in the human isolated gastric fundus was explored.By using the nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-diaphorase and specific rabbit polyclonal NO-synthase (NOS) and VIP antisera, NOS- and VIP-containing varicose nerve fibres were identified throughout the muscle layer or wrapping ganglion cell bodies of the myenteric plexus. NOS-immunoreactive (IR) neural cell bodies were more abundant than those positive for VIP-IR. The majority of myenteric neurones containing VIP coexpressed NADPH-diaphorase.Electrical stimulation of fundus strips caused frequency-dependent NANC relaxations. NG-nitro-L-arginine (L-NOARG: 300 μM) enhanced the basal tone, abolished relaxations to 0.3–3 Hz (5 s) and those to 1 Hz (5 min), markedly reduced (∼50%) those elicited by 10–50 Hz, and unmasked or potentiated excitatory cholinergic responses at frequencies 1 Hz. L-NOARG-resistant relaxations were virtually abolished by VIP (100 nM) desensitization at all frequencies.Relaxations to graded low mechanical distension (1 g) were insensitive to tetrodotoxin (TTX: 1 μM) and L-NOARG (300 μM), while those to higher distensions (2 g) were slightly inhibited by both agents to the same extent (∼25%).In the human gastric fundus, NOS- and VIP immunoreactivities are colocalized in the majority of myenteric neurones. NO and VIP mediate electrically evoked relaxations: low frequency stimulation, irrespective of the duration, caused NO release only, whereas shortlasting stimulation at high frequencies induced NO and VIP release. Relaxations to graded mechanical distension were mostly due to passive viscoelastic properties, with a slight NO-mediated neurogenic component at 2 g distension. The difference between NO and VIP release suggests that in human fundus accommodation is initiated by NO.British Journal of Pharmacology (2000) 129, 12–20; doi:10.1038/sj.bjp.0702977
Experiments were carried out in human detrusor strips to characterize muscarinic receptor subtype... more Experiments were carried out in human detrusor strips to characterize muscarinic receptor subtypes involved in the prejunctional regulation of acetylcholine (ACh) release from cholinergic nerve terminals, and in the postjunctional smooth muscle contractile response.In detrusor strips preincubated with [3H]-choline, electrical field stimulation (600 pulses) delivered in six trains at 10 Hz produced a tritium outflow and a contractile response. In the presence of 10 μM paraoxon (to prevent ACh degradation) the tritium outflow was characterized by HPLC analysis as [3H]-ACh (76%) and [3H]-choline (24%).Electrically-evoked [3H]-ACh release was abolished by tetrodotoxin (TTX: 300 nM) and unaffected by hexamethonium (10 μM), indicating a postganglionic event. It was reduced by physostigmine (100 nM) and the muscarinic receptor agonist, muscarone (10 nM–1 μM), and enhanced by atropine (0.1–100 nM). These findings indicate the presence of a muscarinic negative feedback mechanism controlling ACh release.The effects of various subtype-preferring muscarinic receptor antagonists were evaluated on [3H]-ACh release and muscle contraction. The rank potency (−log EC50) orders at pre- and postjunctional level were: atropine 4-diphenyl-acetoxy-N-piperidine (4-DAMP)>mamba toxin 3 (MT-3)>tripitramine>para-fluorohexahydrosiladiphenidol (pF-HHSiD)methoctraminepirenzepine>tripinamide, and atropine4-DAMP>pF-HHSiD>>pirenzepine=tripitramine>tripinamide>methoctramine>>MT-3, respectively.The comparison of pre- and post-junctional potencies and the relationship analysis with the affinity constants at human cloned muscarinic receptor subtypes indicates that the muscarinic autoreceptor inhibiting ACh release in human detrusor is an M4 receptor, while the receptor involved in muscular contraction belongs to the M3 subtype.Experiments were carried out in human detrusor strips to characterize muscarinic receptor subtypes involved in the prejunctional regulation of acetylcholine (ACh) release from cholinergic nerve terminals, and in the postjunctional smooth muscle contractile response.In detrusor strips preincubated with [3H]-choline, electrical field stimulation (600 pulses) delivered in six trains at 10 Hz produced a tritium outflow and a contractile response. In the presence of 10 μM paraoxon (to prevent ACh degradation) the tritium outflow was characterized by HPLC analysis as [3H]-ACh (76%) and [3H]-choline (24%).Electrically-evoked [3H]-ACh release was abolished by tetrodotoxin (TTX: 300 nM) and unaffected by hexamethonium (10 μM), indicating a postganglionic event. It was reduced by physostigmine (100 nM) and the muscarinic receptor agonist, muscarone (10 nM–1 μM), and enhanced by atropine (0.1–100 nM). These findings indicate the presence of a muscarinic negative feedback mechanism controlling ACh release.The effects of various subtype-preferring muscarinic receptor antagonists were evaluated on [3H]-ACh release and muscle contraction. The rank potency (−log EC50) orders at pre- and postjunctional level were: atropine 4-diphenyl-acetoxy-N-piperidine (4-DAMP)>mamba toxin 3 (MT-3)>tripitramine>para-fluorohexahydrosiladiphenidol (pF-HHSiD)methoctraminepirenzepine>tripinamide, and atropine4-DAMP>pF-HHSiD>>pirenzepine=tripitramine>tripinamide>methoctramine>>MT-3, respectively.The comparison of pre- and post-junctional potencies and the relationship analysis with the affinity constants at human cloned muscarinic receptor subtypes indicates that the muscarinic autoreceptor inhibiting ACh release in human detrusor is an M4 receptor, while the receptor involved in muscular contraction belongs to the M3 subtype.British Journal of Pharmacology (2000) 129, 493–500; doi:10.1038/sj.bjp.0703080
Abstract: The effects of norfloxacin and enoxacin were examined on spontaneous motor activity in ... more Abstract: The effects of norfloxacin and enoxacin were examined on spontaneous motor activity in the guinea-pig isolated ileum. Micromolar concentrations of both compounds caused a biphasic response consisting of relaxation followed by transient contraction. Relaxation to norfloxacin, which was unaffected by phentolamine, propranolol and hyoscine (each at 1 μM), was partially sensitive to tetrodotoxin (1 μM). This indicates that the response is partly mediated by non-adrenergic non-cholinergic (NANC) inhibitory nerves, and partly related to a direct action on the smooth muscle. Apamin (0.1 μM) and suramin (300 μM) inhibited norfloxacin-induced relaxations to an extent similar to that of tetrodotoxin. Conversely, NG-nitro-L-arginine (300 μM) was ineffective. In the presence of theophylline (100 μM) and 3-isobutyl-1-methylxanthine (10 μM), norfloxacin caused relaxation less effective than when added alone. Based on this observation, the NANC component of the relaxation apparently depends on ATP release, whereas the direct component might be due, at least in part, to phosphodiesterase inhibition. Norfloxacin-induced contractions were neurogenic and cholinergic in nature. They were reduced by indomethacin or S-ketoprofen (both at 0.01–1 μM) and suramin (300 μM), suggesting involvement of local prostaglandin production probably induced by ATP release. Previous findings revealed that norfloxacin acted as a non-competitive antagonist at enteric GABAA receptors. In this study the same property was shared by enoxacin against the contractile response to 3-aminopropane sulphonic acid (3-APS), a GABAA receptor agonist. In conclusion, fluoroquinolones exert inhibitory and excitatory effects in the guinea-pig ileum. These are mediated by ATP, prostaglandin and acetylcholine release that might underlie, at least in part, the alterations of gastrointestinal motility observed after fluoroquinolone administration. Furthermore, isolated intestinal preparations might be useful to predict the GABAA-antagonist potential of this class of compounds.
Naunyn-schmiedebergs Archives of Pharmacology, 1998
The novel opioid tetrapeptides, endomorphin-1 and endomorphin-2, recently isolated from bovine an... more The novel opioid tetrapeptides, endomorphin-1 and endomorphin-2, recently isolated from bovine and human brain bind with high affinity and selectivity to central µ-opioid receptors. In the digestive tract, a comprehensive pharmacological analysis of the receptors involved in endomorphin action has not been reported. In this study, we analyzed the effects of endomorphin-1 and endomorphin-2 on longitudinal muscle-myenteric plexus preparations (LMMPs) from the guinea-pig ileum. Both peptides (30 pM–1 µM) inhibited (–log EC50 values: 8.61 and 8.59, respectively) the amplitude of electrically-induced twitch contractions in a concentration-dependent fashion, up to its abolition. Conversely, in unstimulated LMMPs, they failed to affect contractions to applied acetylcholine (100 nM). In stimulated LMMPs, the highly selective µ-opioid receptor antagonist, d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), caused a concentration-dependent (30 nM–1 µM), parallel rightward shift of endomorphin-1 and endomorphin-2 inhibitory curves, without depression of their maximum. Following Schild analysis, calculated pA 2 values were 7.81 and 7.85, respectively, with slopes not different from unity. Concentration-response curves to both peptides were not affected by 30 nM naltrindole (a selective δ-receptor antagonist) or 30 nM nor-binaltorphimine (a selective κ-receptor antagonist). These results demonstrate that endomorphins selectively activate µ-opioid receptors located on excitatory myenteric plexus neurons, and that they act as full agonists.
Background. The dopamine D receptor antagonist levosulpiride is a substituted benzamide derivativ... more Background. The dopamine D receptor antagonist levosulpiride is a substituted benzamide derivative, whose gastrokinetic properties 2 are exploited clinically for the management of functional dyspepsia. However, for other benzamide derivatives, such as cisapride and mosapride, agonism towards serotonin 5-HT receptors is considered the main mechanism leading to gastrointestinal prokinesia. 4 Aims. To assess whether levosulpiride is able to activate 5-HT receptors in the guinea-pig isolated gastrointestinal tract. 4 Materials and methods. Circular muscle strips from gastric antrum, and colonic longitudinal muscle strips were used to detect electrically stimulated neurogenic contractions. The effect of levosulpiride was assessed in the absence and presence of GR125487, a selective 5-HT receptor antagonist. Furthermore, potential interaction of levosulpiride with 5-HT receptors and tissue cholinesterases 4 3 was assessed in unstimulated ileal longitudinal muscle-myenteric plexus preparations. Results. Antral and colonic strip contractions were cholinergic / tachykinergic in nature. Micromolar concentrations of levosulpiride potentiated submaximal responses, through a mechanism competitively antagonized by GR125487 (pK 59.4). In LMMPs, levosulpiride B slightly affected contractions caused by the 5-HT receptor agonist 2-methyl-5-HT, and had no effect on contractions to exogenous 3 acetylcholine.
The effect of the melatonin receptor ligand, 2-phenylmelatonin, has been assessed in isolated str... more The effect of the melatonin receptor ligand, 2-phenylmelatonin, has been assessed in isolated strips of the guinea-pig proximal colon. 2-Phenylmelatonin (0.01 nM-1 μM) caused a concentration-dependent contractile response. The potency value (−log EC50) was 9.3±1.0. The maximum effect was 25±4% of that elicited by the maximally effective concentration (0.3 μM) of 5-HT and 43±3% of that by the maximally effective concentration (10 μM) of melatonin. When used as an antagonist, 2-phenylmelatonin (0.01 nM and 0.1 nM) concentration-dependently inhibited melatonin-induced contractions with depression of the maximum response by 25% and 54%, respectively. Higher (1 nM) 2-phenylmelatonin concentrations failed to antagonize melatonin-induced response. Prazosin (0.3 μM), a selective antagonist of melatonin MT3 sites, antagonized melatonin-induced contractions to an extent similar to that induced by 0.01 nM 2-phenylmelatonin (with 30% reduction of the maximum effect to melatonin). The combination of 0.3 μM prazosin and 0.01 nM 2-phenylmelatonin caused antagonism similar in extent to that caused by each individual antagonist. 2-Phenylmelatonin at subnanomolar concentrations behaves as an antagonist of melatonin-induced contractile responses while at nanomolar/micromolar concentrations it behaves as a weak contractile agonist.
The effect of the melatonin receptor ligand, 2-phenylmelatonin, has been assessed in isolated str... more The effect of the melatonin receptor ligand, 2-phenylmelatonin, has been assessed in isolated strips of the guineapig proximal colon. 2-Phenylmelatonin (0.01 nM-1 mM) caused a concentration-dependent contractile response. The potency value (ªlog EC 50 ) was 9.3∫1.0. The maximum effect was 25∫4% of that elicited by the maximally effective concentration (0.3 mM) of 5-HT and 43∫3% of that by the maximally effective concentration (10 mM) of melatonin. When used as an antagonist, 2-phenylmelatonin (0.01 nM and 0.1 nM) concentration-dependently inhibited melatonin-induced contractions with depression of the maximum response by 25% and 54%, respectively. Higher (1 nM) 2-phenylmelatonin concentrations failed to antagonize melatonin-induced response. Prazosin (0.3 mM), a selective antagonist of melatonin MT 3 sites, antagonized melatonin-induced contractions to an extent similar to that induced by 0.01 nM 2-phenylmelatonin (with 30% reduction of the maximum effect to melatonin). The combination of 0.3 mM prazosin and 0.01 nM 2-phenylmelatonin caused antagonism similar in extent to that caused by each individual antagonist. 2-Phenylmelatonin at subnanomolar concentrations behaves as an antagonist of melatonin-induced contractile responses while at nanomolar/micromolar concentrations it behaves as a weak contractile agonist.
The morphological pattern and motor correlates of nitric oxide (NO) and vasoactive intestinal pol... more The morphological pattern and motor correlates of nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) innervation in the human isolated gastric fundus was explored.By using the nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-diaphorase and specific rabbit polyclonal NO-synthase (NOS) and VIP antisera, NOS- and VIP-containing varicose nerve fibres were identified throughout the muscle layer or wrapping ganglion cell bodies of the myenteric plexus. NOS-immunoreactive (IR) neural cell bodies were more abundant than those positive for VIP-IR. The majority of myenteric neurones containing VIP coexpressed NADPH-diaphorase.Electrical stimulation of fundus strips caused frequency-dependent NANC relaxations. NG-nitro-L-arginine (L-NOARG: 300 μM) enhanced the basal tone, abolished relaxations to 0.3–3 Hz (5 s) and those to 1 Hz (5 min), markedly reduced (∼50%) those elicited by 10–50 Hz, and unmasked or potentiated excitatory cholinergic responses at frequencies 1 Hz. L-NOARG-resistant relaxations were virtually abolished by VIP (100 nM) desensitization at all frequencies.Relaxations to graded low mechanical distension (1 g) were insensitive to tetrodotoxin (TTX: 1 μM) and L-NOARG (300 μM), while those to higher distensions (2 g) were slightly inhibited by both agents to the same extent (∼25%).In the human gastric fundus, NOS- and VIP immunoreactivities are colocalized in the majority of myenteric neurones. NO and VIP mediate electrically evoked relaxations: low frequency stimulation, irrespective of the duration, caused NO release only, whereas shortlasting stimulation at high frequencies induced NO and VIP release. Relaxations to graded mechanical distension were mostly due to passive viscoelastic properties, with a slight NO-mediated neurogenic component at 2 g distension. The difference between NO and VIP release suggests that in human fundus accommodation is initiated by NO.The morphological pattern and motor correlates of nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) innervation in the human isolated gastric fundus was explored.By using the nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-diaphorase and specific rabbit polyclonal NO-synthase (NOS) and VIP antisera, NOS- and VIP-containing varicose nerve fibres were identified throughout the muscle layer or wrapping ganglion cell bodies of the myenteric plexus. NOS-immunoreactive (IR) neural cell bodies were more abundant than those positive for VIP-IR. The majority of myenteric neurones containing VIP coexpressed NADPH-diaphorase.Electrical stimulation of fundus strips caused frequency-dependent NANC relaxations. NG-nitro-L-arginine (L-NOARG: 300 μM) enhanced the basal tone, abolished relaxations to 0.3–3 Hz (5 s) and those to 1 Hz (5 min), markedly reduced (∼50%) those elicited by 10–50 Hz, and unmasked or potentiated excitatory cholinergic responses at frequencies 1 Hz. L-NOARG-resistant relaxations were virtually abolished by VIP (100 nM) desensitization at all frequencies.Relaxations to graded low mechanical distension (1 g) were insensitive to tetrodotoxin (TTX: 1 μM) and L-NOARG (300 μM), while those to higher distensions (2 g) were slightly inhibited by both agents to the same extent (∼25%).In the human gastric fundus, NOS- and VIP immunoreactivities are colocalized in the majority of myenteric neurones. NO and VIP mediate electrically evoked relaxations: low frequency stimulation, irrespective of the duration, caused NO release only, whereas shortlasting stimulation at high frequencies induced NO and VIP release. Relaxations to graded mechanical distension were mostly due to passive viscoelastic properties, with a slight NO-mediated neurogenic component at 2 g distension. The difference between NO and VIP release suggests that in human fundus accommodation is initiated by NO.British Journal of Pharmacology (2000) 129, 12–20; doi:10.1038/sj.bjp.0702977
Experiments were carried out in human detrusor strips to characterize muscarinic receptor subtype... more Experiments were carried out in human detrusor strips to characterize muscarinic receptor subtypes involved in the prejunctional regulation of acetylcholine (ACh) release from cholinergic nerve terminals, and in the postjunctional smooth muscle contractile response.In detrusor strips preincubated with [3H]-choline, electrical field stimulation (600 pulses) delivered in six trains at 10 Hz produced a tritium outflow and a contractile response. In the presence of 10 μM paraoxon (to prevent ACh degradation) the tritium outflow was characterized by HPLC analysis as [3H]-ACh (76%) and [3H]-choline (24%).Electrically-evoked [3H]-ACh release was abolished by tetrodotoxin (TTX: 300 nM) and unaffected by hexamethonium (10 μM), indicating a postganglionic event. It was reduced by physostigmine (100 nM) and the muscarinic receptor agonist, muscarone (10 nM–1 μM), and enhanced by atropine (0.1–100 nM). These findings indicate the presence of a muscarinic negative feedback mechanism controlling ACh release.The effects of various subtype-preferring muscarinic receptor antagonists were evaluated on [3H]-ACh release and muscle contraction. The rank potency (−log EC50) orders at pre- and postjunctional level were: atropine 4-diphenyl-acetoxy-N-piperidine (4-DAMP)>mamba toxin 3 (MT-3)>tripitramine>para-fluorohexahydrosiladiphenidol (pF-HHSiD)methoctraminepirenzepine>tripinamide, and atropine4-DAMP>pF-HHSiD>>pirenzepine=tripitramine>tripinamide>methoctramine>>MT-3, respectively.The comparison of pre- and post-junctional potencies and the relationship analysis with the affinity constants at human cloned muscarinic receptor subtypes indicates that the muscarinic autoreceptor inhibiting ACh release in human detrusor is an M4 receptor, while the receptor involved in muscular contraction belongs to the M3 subtype.Experiments were carried out in human detrusor strips to characterize muscarinic receptor subtypes involved in the prejunctional regulation of acetylcholine (ACh) release from cholinergic nerve terminals, and in the postjunctional smooth muscle contractile response.In detrusor strips preincubated with [3H]-choline, electrical field stimulation (600 pulses) delivered in six trains at 10 Hz produced a tritium outflow and a contractile response. In the presence of 10 μM paraoxon (to prevent ACh degradation) the tritium outflow was characterized by HPLC analysis as [3H]-ACh (76%) and [3H]-choline (24%).Electrically-evoked [3H]-ACh release was abolished by tetrodotoxin (TTX: 300 nM) and unaffected by hexamethonium (10 μM), indicating a postganglionic event. It was reduced by physostigmine (100 nM) and the muscarinic receptor agonist, muscarone (10 nM–1 μM), and enhanced by atropine (0.1–100 nM). These findings indicate the presence of a muscarinic negative feedback mechanism controlling ACh release.The effects of various subtype-preferring muscarinic receptor antagonists were evaluated on [3H]-ACh release and muscle contraction. The rank potency (−log EC50) orders at pre- and postjunctional level were: atropine 4-diphenyl-acetoxy-N-piperidine (4-DAMP)>mamba toxin 3 (MT-3)>tripitramine>para-fluorohexahydrosiladiphenidol (pF-HHSiD)methoctraminepirenzepine>tripinamide, and atropine4-DAMP>pF-HHSiD>>pirenzepine=tripitramine>tripinamide>methoctramine>>MT-3, respectively.The comparison of pre- and post-junctional potencies and the relationship analysis with the affinity constants at human cloned muscarinic receptor subtypes indicates that the muscarinic autoreceptor inhibiting ACh release in human detrusor is an M4 receptor, while the receptor involved in muscular contraction belongs to the M3 subtype.British Journal of Pharmacology (2000) 129, 493–500; doi:10.1038/sj.bjp.0703080
1 Experiments were carried out in human detrusor strips to characterize muscarinic receptor subty... more 1 Experiments were carried out in human detrusor strips to characterize muscarinic receptor subtypes involved in the prejunctional regulation of acetylcholine (ACh) release from cholinergic nerve terminals, and in the postjunctional smooth muscle contractile response. 2 In detrusor strips preincubated with [ 3 H]-choline, electrical ®eld stimulation (600 pulses) delivered in six trains at 10 Hz produced a tritium out¯ow and a contractile response. In the presence of 10 mM paraoxon (to prevent ACh degradation) the tritium out¯ow was characterized by HPLC analysis as [ 3 H]-ACh (76%) and [ 3 H]-choline (24%).
The effects of norlloxacin and enoxacin were examined on spontaneous motor activity in the guinea... more The effects of norlloxacin and enoxacin were examined on spontaneous motor activity in the guinea-pig isolated ileum. Micromolar concentrations of both compounds caused a biphasic response consisting of relaxation followed by transient contraction. Relaxation to noriloxacin, which was unaffected by phentolamine, propranolol and hyoscine (each at 1 pM), was partially sensitive to tetrodotoxin (1 pM). This indicates that the response is partly mediated by nonadrenergic non-cholinergic (NANC) inhibitory nerves, and partly related to a direct action on the smooth muscle. Apamin (0.1 pM) and suramin (300 pM) inhibited norfloxacin-induced relaxations to an extent similar to that of tetrodotoxin. Conversely, NG-nitro-L-arginine (300 pM) was ineffective. In the presence of theophylline (100 pM) and 3-isobutyl-lmethylxanthine (1 0 pM), norfloxacin caused relaxation less effective than when added alone. Based on this observation, the NANC component of the relaxation apparently depends on ATP release, whereas the direct component might be due, at least in part, to phosphodiesterase inhibition. Norfloxacin-induced contractions were neurogenic and cholinergic in nature. They were reduced by indomethacin or S-ketoprofen (both at 0.01-1 pM) and suramin (300 pM), suggesting involvement of local prostaglandin production probably induced by ATP release. Previous findings revealed that norfloxacin acted as a non-competitive antagonist at enteric GABAA receptors. In this study the same property was shared by enoxacin against the contractile response to 3-aminopropane sulphonic acid (3-APS), a GABAA receptor agonist. In conclusion, fluoroquinolones exert inhibitory and excitatory effects in the guinea-pig ileum. These are mediated by ATE prostaglandin and acetylcholine release that might underlie, at least in part, the alterations of gastrointestinal motility observed after fluoroquinolone administration. Furthermore, isolated intestinal preparations might be useful to predict the GABAA-antagonist potential of this class of compounds.
Ischemic pain-examples include the chest pain of a heart attack and the leg pain of a 30 s sprint... more Ischemic pain-examples include the chest pain of a heart attack and the leg pain of a 30 s sprint-occurs when muscle gets too little oxygen for its metabolic need. Lactic acid cannot act alone to trigger ischemic pain because the pH change is so small. Here, we show that another compound released from ischemic muscle, adenosine tri-phosphate (ATP), works together with acid by increasing the pH sensitivity of acid-sensing ion channel number 3 (ASIC3), the molecule used by sensory neurons to detect lactic acidosis. Our data argue that ATP acts by binding to P2X receptors that form a molecular complex with ASICs; the receptor on sensory neurons appears to be P2X5, an electrically quiet ion channel. Coincident detection of acid and ATP should confer sensory selectivity for ischemia over other conditions of acidosis.
Abstract: The effects of norfloxacin and enoxacin were examined on spontaneous motor activity in ... more Abstract: The effects of norfloxacin and enoxacin were examined on spontaneous motor activity in the guinea-pig isolated ileum. Micromolar concentrations of both compounds caused a biphasic response consisting of relaxation followed by transient contraction. Relaxation to norfloxacin, which was unaffected by phentolamine, propranolol and hyoscine (each at 1 μM), was partially sensitive to tetrodotoxin (1 μM). This indicates that the response is partly mediated by non-adrenergic non-cholinergic (NANC) inhibitory nerves, and partly related to a direct action on the smooth muscle. Apamin (0.1 μM) and suramin (300 μM) inhibited norfloxacin-induced relaxations to an extent similar to that of tetrodotoxin. Conversely, NG-nitro-L-arginine (300 μM) was ineffective. In the presence of theophylline (100 μM) and 3-isobutyl-1-methylxanthine (10 μM), norfloxacin caused relaxation less effective than when added alone. Based on this observation, the NANC component of the relaxation apparently depends on ATP release, whereas the direct component might be due, at least in part, to phosphodiesterase inhibition. Norfloxacin-induced contractions were neurogenic and cholinergic in nature. They were reduced by indomethacin or S-ketoprofen (both at 0.01–1 μM) and suramin (300 μM), suggesting involvement of local prostaglandin production probably induced by ATP release. Previous findings revealed that norfloxacin acted as a non-competitive antagonist at enteric GABAA receptors. In this study the same property was shared by enoxacin against the contractile response to 3-aminopropane sulphonic acid (3-APS), a GABAA receptor agonist. In conclusion, fluoroquinolones exert inhibitory and excitatory effects in the guinea-pig ileum. These are mediated by ATP, prostaglandin and acetylcholine release that might underlie, at least in part, the alterations of gastrointestinal motility observed after fluoroquinolone administration. Furthermore, isolated intestinal preparations might be useful to predict the GABAA-antagonist potential of this class of compounds.
The effect of the melatonin receptor ligand, 2-phenylmelatonin, has been assessed in isolated str... more The effect of the melatonin receptor ligand, 2-phenylmelatonin, has been assessed in isolated strips of the guinea-pig proximal colon. 2-Phenylmelatonin (0.01 nM-1 μM) caused a concentration-dependent contractile response. The potency value (−log EC50) was 9.3±1.0. The maximum effect was 25±4% of that elicited by the maximally effective concentration (0.3 μM) of 5-HT and 43±3% of that by the maximally effective concentration (10 μM) of melatonin. When used as an antagonist, 2-phenylmelatonin (0.01 nM and 0.1 nM) concentration-dependently inhibited melatonin-induced contractions with depression of the maximum response by 25% and 54%, respectively. Higher (1 nM) 2-phenylmelatonin concentrations failed to antagonize melatonin-induced response. Prazosin (0.3 μM), a selective antagonist of melatonin MT3 sites, antagonized melatonin-induced contractions to an extent similar to that induced by 0.01 nM 2-phenylmelatonin (with 30% reduction of the maximum effect to melatonin). The combination of 0.3 μM prazosin and 0.01 nM 2-phenylmelatonin caused antagonism similar in extent to that caused by each individual antagonist. 2-Phenylmelatonin at subnanomolar concentrations behaves as an antagonist of melatonin-induced contractile responses while at nanomolar/micromolar concentrations it behaves as a weak contractile agonist.
The effect of the melatonin receptor ligand, 2-phenylmelatonin, has been assessed in isolated str... more The effect of the melatonin receptor ligand, 2-phenylmelatonin, has been assessed in isolated strips of the guineapig proximal colon. 2-Phenylmelatonin (0.01 nM-1 mM) caused a concentration-dependent contractile response. The potency value (ªlog EC 50 ) was 9.3∫1.0. The maximum effect was 25∫4% of that elicited by the maximally effective concentration (0.3 mM) of 5-HT and 43∫3% of that by the maximally effective concentration (10 mM) of melatonin. When used as an antagonist, 2-phenylmelatonin (0.01 nM and 0.1 nM) concentration-dependently inhibited melatonin-induced contractions with depression of the maximum response by 25% and 54%, respectively. Higher (1 nM) 2-phenylmelatonin concentrations failed to antagonize melatonin-induced response. Prazosin (0.3 mM), a selective antagonist of melatonin MT 3 sites, antagonized melatonin-induced contractions to an extent similar to that induced by 0.01 nM 2-phenylmelatonin (with 30% reduction of the maximum effect to melatonin). The combination of 0.3 mM prazosin and 0.01 nM 2-phenylmelatonin caused antagonism similar in extent to that caused by each individual antagonist. 2-Phenylmelatonin at subnanomolar concentrations behaves as an antagonist of melatonin-induced contractile responses while at nanomolar/micromolar concentrations it behaves as a weak contractile agonist.
The morphological pattern and motor correlates of nitric oxide (NO) and vasoactive intestinal pol... more The morphological pattern and motor correlates of nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) innervation in the human isolated gastric fundus was explored.By using the nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-diaphorase and specific rabbit polyclonal NO-synthase (NOS) and VIP antisera, NOS- and VIP-containing varicose nerve fibres were identified throughout the muscle layer or wrapping ganglion cell bodies of the myenteric plexus. NOS-immunoreactive (IR) neural cell bodies were more abundant than those positive for VIP-IR. The majority of myenteric neurones containing VIP coexpressed NADPH-diaphorase.Electrical stimulation of fundus strips caused frequency-dependent NANC relaxations. NG-nitro-L-arginine (L-NOARG: 300 μM) enhanced the basal tone, abolished relaxations to 0.3–3 Hz (5 s) and those to 1 Hz (5 min), markedly reduced (∼50%) those elicited by 10–50 Hz, and unmasked or potentiated excitatory cholinergic responses at frequencies 1 Hz. L-NOARG-resistant relaxations were virtually abolished by VIP (100 nM) desensitization at all frequencies.Relaxations to graded low mechanical distension (1 g) were insensitive to tetrodotoxin (TTX: 1 μM) and L-NOARG (300 μM), while those to higher distensions (2 g) were slightly inhibited by both agents to the same extent (∼25%).In the human gastric fundus, NOS- and VIP immunoreactivities are colocalized in the majority of myenteric neurones. NO and VIP mediate electrically evoked relaxations: low frequency stimulation, irrespective of the duration, caused NO release only, whereas shortlasting stimulation at high frequencies induced NO and VIP release. Relaxations to graded mechanical distension were mostly due to passive viscoelastic properties, with a slight NO-mediated neurogenic component at 2 g distension. The difference between NO and VIP release suggests that in human fundus accommodation is initiated by NO.The morphological pattern and motor correlates of nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) innervation in the human isolated gastric fundus was explored.By using the nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-diaphorase and specific rabbit polyclonal NO-synthase (NOS) and VIP antisera, NOS- and VIP-containing varicose nerve fibres were identified throughout the muscle layer or wrapping ganglion cell bodies of the myenteric plexus. NOS-immunoreactive (IR) neural cell bodies were more abundant than those positive for VIP-IR. The majority of myenteric neurones containing VIP coexpressed NADPH-diaphorase.Electrical stimulation of fundus strips caused frequency-dependent NANC relaxations. NG-nitro-L-arginine (L-NOARG: 300 μM) enhanced the basal tone, abolished relaxations to 0.3–3 Hz (5 s) and those to 1 Hz (5 min), markedly reduced (∼50%) those elicited by 10–50 Hz, and unmasked or potentiated excitatory cholinergic responses at frequencies 1 Hz. L-NOARG-resistant relaxations were virtually abolished by VIP (100 nM) desensitization at all frequencies.Relaxations to graded low mechanical distension (1 g) were insensitive to tetrodotoxin (TTX: 1 μM) and L-NOARG (300 μM), while those to higher distensions (2 g) were slightly inhibited by both agents to the same extent (∼25%).In the human gastric fundus, NOS- and VIP immunoreactivities are colocalized in the majority of myenteric neurones. NO and VIP mediate electrically evoked relaxations: low frequency stimulation, irrespective of the duration, caused NO release only, whereas shortlasting stimulation at high frequencies induced NO and VIP release. Relaxations to graded mechanical distension were mostly due to passive viscoelastic properties, with a slight NO-mediated neurogenic component at 2 g distension. The difference between NO and VIP release suggests that in human fundus accommodation is initiated by NO.British Journal of Pharmacology (2000) 129, 12–20; doi:10.1038/sj.bjp.0702977
Experiments were carried out in human detrusor strips to characterize muscarinic receptor subtype... more Experiments were carried out in human detrusor strips to characterize muscarinic receptor subtypes involved in the prejunctional regulation of acetylcholine (ACh) release from cholinergic nerve terminals, and in the postjunctional smooth muscle contractile response.In detrusor strips preincubated with [3H]-choline, electrical field stimulation (600 pulses) delivered in six trains at 10 Hz produced a tritium outflow and a contractile response. In the presence of 10 μM paraoxon (to prevent ACh degradation) the tritium outflow was characterized by HPLC analysis as [3H]-ACh (76%) and [3H]-choline (24%).Electrically-evoked [3H]-ACh release was abolished by tetrodotoxin (TTX: 300 nM) and unaffected by hexamethonium (10 μM), indicating a postganglionic event. It was reduced by physostigmine (100 nM) and the muscarinic receptor agonist, muscarone (10 nM–1 μM), and enhanced by atropine (0.1–100 nM). These findings indicate the presence of a muscarinic negative feedback mechanism controlling ACh release.The effects of various subtype-preferring muscarinic receptor antagonists were evaluated on [3H]-ACh release and muscle contraction. The rank potency (−log EC50) orders at pre- and postjunctional level were: atropine 4-diphenyl-acetoxy-N-piperidine (4-DAMP)>mamba toxin 3 (MT-3)>tripitramine>para-fluorohexahydrosiladiphenidol (pF-HHSiD)methoctraminepirenzepine>tripinamide, and atropine4-DAMP>pF-HHSiD>>pirenzepine=tripitramine>tripinamide>methoctramine>>MT-3, respectively.The comparison of pre- and post-junctional potencies and the relationship analysis with the affinity constants at human cloned muscarinic receptor subtypes indicates that the muscarinic autoreceptor inhibiting ACh release in human detrusor is an M4 receptor, while the receptor involved in muscular contraction belongs to the M3 subtype.Experiments were carried out in human detrusor strips to characterize muscarinic receptor subtypes involved in the prejunctional regulation of acetylcholine (ACh) release from cholinergic nerve terminals, and in the postjunctional smooth muscle contractile response.In detrusor strips preincubated with [3H]-choline, electrical field stimulation (600 pulses) delivered in six trains at 10 Hz produced a tritium outflow and a contractile response. In the presence of 10 μM paraoxon (to prevent ACh degradation) the tritium outflow was characterized by HPLC analysis as [3H]-ACh (76%) and [3H]-choline (24%).Electrically-evoked [3H]-ACh release was abolished by tetrodotoxin (TTX: 300 nM) and unaffected by hexamethonium (10 μM), indicating a postganglionic event. It was reduced by physostigmine (100 nM) and the muscarinic receptor agonist, muscarone (10 nM–1 μM), and enhanced by atropine (0.1–100 nM). These findings indicate the presence of a muscarinic negative feedback mechanism controlling ACh release.The effects of various subtype-preferring muscarinic receptor antagonists were evaluated on [3H]-ACh release and muscle contraction. The rank potency (−log EC50) orders at pre- and postjunctional level were: atropine 4-diphenyl-acetoxy-N-piperidine (4-DAMP)>mamba toxin 3 (MT-3)>tripitramine>para-fluorohexahydrosiladiphenidol (pF-HHSiD)methoctraminepirenzepine>tripinamide, and atropine4-DAMP>pF-HHSiD>>pirenzepine=tripitramine>tripinamide>methoctramine>>MT-3, respectively.The comparison of pre- and post-junctional potencies and the relationship analysis with the affinity constants at human cloned muscarinic receptor subtypes indicates that the muscarinic autoreceptor inhibiting ACh release in human detrusor is an M4 receptor, while the receptor involved in muscular contraction belongs to the M3 subtype.British Journal of Pharmacology (2000) 129, 493–500; doi:10.1038/sj.bjp.0703080
Abstract: The effects of norfloxacin and enoxacin were examined on spontaneous motor activity in ... more Abstract: The effects of norfloxacin and enoxacin were examined on spontaneous motor activity in the guinea-pig isolated ileum. Micromolar concentrations of both compounds caused a biphasic response consisting of relaxation followed by transient contraction. Relaxation to norfloxacin, which was unaffected by phentolamine, propranolol and hyoscine (each at 1 μM), was partially sensitive to tetrodotoxin (1 μM). This indicates that the response is partly mediated by non-adrenergic non-cholinergic (NANC) inhibitory nerves, and partly related to a direct action on the smooth muscle. Apamin (0.1 μM) and suramin (300 μM) inhibited norfloxacin-induced relaxations to an extent similar to that of tetrodotoxin. Conversely, NG-nitro-L-arginine (300 μM) was ineffective. In the presence of theophylline (100 μM) and 3-isobutyl-1-methylxanthine (10 μM), norfloxacin caused relaxation less effective than when added alone. Based on this observation, the NANC component of the relaxation apparently depends on ATP release, whereas the direct component might be due, at least in part, to phosphodiesterase inhibition. Norfloxacin-induced contractions were neurogenic and cholinergic in nature. They were reduced by indomethacin or S-ketoprofen (both at 0.01–1 μM) and suramin (300 μM), suggesting involvement of local prostaglandin production probably induced by ATP release. Previous findings revealed that norfloxacin acted as a non-competitive antagonist at enteric GABAA receptors. In this study the same property was shared by enoxacin against the contractile response to 3-aminopropane sulphonic acid (3-APS), a GABAA receptor agonist. In conclusion, fluoroquinolones exert inhibitory and excitatory effects in the guinea-pig ileum. These are mediated by ATP, prostaglandin and acetylcholine release that might underlie, at least in part, the alterations of gastrointestinal motility observed after fluoroquinolone administration. Furthermore, isolated intestinal preparations might be useful to predict the GABAA-antagonist potential of this class of compounds.
Naunyn-schmiedebergs Archives of Pharmacology, 1998
The novel opioid tetrapeptides, endomorphin-1 and endomorphin-2, recently isolated from bovine an... more The novel opioid tetrapeptides, endomorphin-1 and endomorphin-2, recently isolated from bovine and human brain bind with high affinity and selectivity to central µ-opioid receptors. In the digestive tract, a comprehensive pharmacological analysis of the receptors involved in endomorphin action has not been reported. In this study, we analyzed the effects of endomorphin-1 and endomorphin-2 on longitudinal muscle-myenteric plexus preparations (LMMPs) from the guinea-pig ileum. Both peptides (30 pM–1 µM) inhibited (–log EC50 values: 8.61 and 8.59, respectively) the amplitude of electrically-induced twitch contractions in a concentration-dependent fashion, up to its abolition. Conversely, in unstimulated LMMPs, they failed to affect contractions to applied acetylcholine (100 nM). In stimulated LMMPs, the highly selective µ-opioid receptor antagonist, d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), caused a concentration-dependent (30 nM–1 µM), parallel rightward shift of endomorphin-1 and endomorphin-2 inhibitory curves, without depression of their maximum. Following Schild analysis, calculated pA 2 values were 7.81 and 7.85, respectively, with slopes not different from unity. Concentration-response curves to both peptides were not affected by 30 nM naltrindole (a selective δ-receptor antagonist) or 30 nM nor-binaltorphimine (a selective κ-receptor antagonist). These results demonstrate that endomorphins selectively activate µ-opioid receptors located on excitatory myenteric plexus neurons, and that they act as full agonists.
Background. The dopamine D receptor antagonist levosulpiride is a substituted benzamide derivativ... more Background. The dopamine D receptor antagonist levosulpiride is a substituted benzamide derivative, whose gastrokinetic properties 2 are exploited clinically for the management of functional dyspepsia. However, for other benzamide derivatives, such as cisapride and mosapride, agonism towards serotonin 5-HT receptors is considered the main mechanism leading to gastrointestinal prokinesia. 4 Aims. To assess whether levosulpiride is able to activate 5-HT receptors in the guinea-pig isolated gastrointestinal tract. 4 Materials and methods. Circular muscle strips from gastric antrum, and colonic longitudinal muscle strips were used to detect electrically stimulated neurogenic contractions. The effect of levosulpiride was assessed in the absence and presence of GR125487, a selective 5-HT receptor antagonist. Furthermore, potential interaction of levosulpiride with 5-HT receptors and tissue cholinesterases 4 3 was assessed in unstimulated ileal longitudinal muscle-myenteric plexus preparations. Results. Antral and colonic strip contractions were cholinergic / tachykinergic in nature. Micromolar concentrations of levosulpiride potentiated submaximal responses, through a mechanism competitively antagonized by GR125487 (pK 59.4). In LMMPs, levosulpiride B slightly affected contractions caused by the 5-HT receptor agonist 2-methyl-5-HT, and had no effect on contractions to exogenous 3 acetylcholine.
The effect of the melatonin receptor ligand, 2-phenylmelatonin, has been assessed in isolated str... more The effect of the melatonin receptor ligand, 2-phenylmelatonin, has been assessed in isolated strips of the guinea-pig proximal colon. 2-Phenylmelatonin (0.01 nM-1 μM) caused a concentration-dependent contractile response. The potency value (−log EC50) was 9.3±1.0. The maximum effect was 25±4% of that elicited by the maximally effective concentration (0.3 μM) of 5-HT and 43±3% of that by the maximally effective concentration (10 μM) of melatonin. When used as an antagonist, 2-phenylmelatonin (0.01 nM and 0.1 nM) concentration-dependently inhibited melatonin-induced contractions with depression of the maximum response by 25% and 54%, respectively. Higher (1 nM) 2-phenylmelatonin concentrations failed to antagonize melatonin-induced response. Prazosin (0.3 μM), a selective antagonist of melatonin MT3 sites, antagonized melatonin-induced contractions to an extent similar to that induced by 0.01 nM 2-phenylmelatonin (with 30% reduction of the maximum effect to melatonin). The combination of 0.3 μM prazosin and 0.01 nM 2-phenylmelatonin caused antagonism similar in extent to that caused by each individual antagonist. 2-Phenylmelatonin at subnanomolar concentrations behaves as an antagonist of melatonin-induced contractile responses while at nanomolar/micromolar concentrations it behaves as a weak contractile agonist.
The effect of the melatonin receptor ligand, 2-phenylmelatonin, has been assessed in isolated str... more The effect of the melatonin receptor ligand, 2-phenylmelatonin, has been assessed in isolated strips of the guineapig proximal colon. 2-Phenylmelatonin (0.01 nM-1 mM) caused a concentration-dependent contractile response. The potency value (ªlog EC 50 ) was 9.3∫1.0. The maximum effect was 25∫4% of that elicited by the maximally effective concentration (0.3 mM) of 5-HT and 43∫3% of that by the maximally effective concentration (10 mM) of melatonin. When used as an antagonist, 2-phenylmelatonin (0.01 nM and 0.1 nM) concentration-dependently inhibited melatonin-induced contractions with depression of the maximum response by 25% and 54%, respectively. Higher (1 nM) 2-phenylmelatonin concentrations failed to antagonize melatonin-induced response. Prazosin (0.3 mM), a selective antagonist of melatonin MT 3 sites, antagonized melatonin-induced contractions to an extent similar to that induced by 0.01 nM 2-phenylmelatonin (with 30% reduction of the maximum effect to melatonin). The combination of 0.3 mM prazosin and 0.01 nM 2-phenylmelatonin caused antagonism similar in extent to that caused by each individual antagonist. 2-Phenylmelatonin at subnanomolar concentrations behaves as an antagonist of melatonin-induced contractile responses while at nanomolar/micromolar concentrations it behaves as a weak contractile agonist.
The morphological pattern and motor correlates of nitric oxide (NO) and vasoactive intestinal pol... more The morphological pattern and motor correlates of nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) innervation in the human isolated gastric fundus was explored.By using the nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-diaphorase and specific rabbit polyclonal NO-synthase (NOS) and VIP antisera, NOS- and VIP-containing varicose nerve fibres were identified throughout the muscle layer or wrapping ganglion cell bodies of the myenteric plexus. NOS-immunoreactive (IR) neural cell bodies were more abundant than those positive for VIP-IR. The majority of myenteric neurones containing VIP coexpressed NADPH-diaphorase.Electrical stimulation of fundus strips caused frequency-dependent NANC relaxations. NG-nitro-L-arginine (L-NOARG: 300 μM) enhanced the basal tone, abolished relaxations to 0.3–3 Hz (5 s) and those to 1 Hz (5 min), markedly reduced (∼50%) those elicited by 10–50 Hz, and unmasked or potentiated excitatory cholinergic responses at frequencies 1 Hz. L-NOARG-resistant relaxations were virtually abolished by VIP (100 nM) desensitization at all frequencies.Relaxations to graded low mechanical distension (1 g) were insensitive to tetrodotoxin (TTX: 1 μM) and L-NOARG (300 μM), while those to higher distensions (2 g) were slightly inhibited by both agents to the same extent (∼25%).In the human gastric fundus, NOS- and VIP immunoreactivities are colocalized in the majority of myenteric neurones. NO and VIP mediate electrically evoked relaxations: low frequency stimulation, irrespective of the duration, caused NO release only, whereas shortlasting stimulation at high frequencies induced NO and VIP release. Relaxations to graded mechanical distension were mostly due to passive viscoelastic properties, with a slight NO-mediated neurogenic component at 2 g distension. The difference between NO and VIP release suggests that in human fundus accommodation is initiated by NO.The morphological pattern and motor correlates of nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) innervation in the human isolated gastric fundus was explored.By using the nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-diaphorase and specific rabbit polyclonal NO-synthase (NOS) and VIP antisera, NOS- and VIP-containing varicose nerve fibres were identified throughout the muscle layer or wrapping ganglion cell bodies of the myenteric plexus. NOS-immunoreactive (IR) neural cell bodies were more abundant than those positive for VIP-IR. The majority of myenteric neurones containing VIP coexpressed NADPH-diaphorase.Electrical stimulation of fundus strips caused frequency-dependent NANC relaxations. NG-nitro-L-arginine (L-NOARG: 300 μM) enhanced the basal tone, abolished relaxations to 0.3–3 Hz (5 s) and those to 1 Hz (5 min), markedly reduced (∼50%) those elicited by 10–50 Hz, and unmasked or potentiated excitatory cholinergic responses at frequencies 1 Hz. L-NOARG-resistant relaxations were virtually abolished by VIP (100 nM) desensitization at all frequencies.Relaxations to graded low mechanical distension (1 g) were insensitive to tetrodotoxin (TTX: 1 μM) and L-NOARG (300 μM), while those to higher distensions (2 g) were slightly inhibited by both agents to the same extent (∼25%).In the human gastric fundus, NOS- and VIP immunoreactivities are colocalized in the majority of myenteric neurones. NO and VIP mediate electrically evoked relaxations: low frequency stimulation, irrespective of the duration, caused NO release only, whereas shortlasting stimulation at high frequencies induced NO and VIP release. Relaxations to graded mechanical distension were mostly due to passive viscoelastic properties, with a slight NO-mediated neurogenic component at 2 g distension. The difference between NO and VIP release suggests that in human fundus accommodation is initiated by NO.British Journal of Pharmacology (2000) 129, 12–20; doi:10.1038/sj.bjp.0702977
Experiments were carried out in human detrusor strips to characterize muscarinic receptor subtype... more Experiments were carried out in human detrusor strips to characterize muscarinic receptor subtypes involved in the prejunctional regulation of acetylcholine (ACh) release from cholinergic nerve terminals, and in the postjunctional smooth muscle contractile response.In detrusor strips preincubated with [3H]-choline, electrical field stimulation (600 pulses) delivered in six trains at 10 Hz produced a tritium outflow and a contractile response. In the presence of 10 μM paraoxon (to prevent ACh degradation) the tritium outflow was characterized by HPLC analysis as [3H]-ACh (76%) and [3H]-choline (24%).Electrically-evoked [3H]-ACh release was abolished by tetrodotoxin (TTX: 300 nM) and unaffected by hexamethonium (10 μM), indicating a postganglionic event. It was reduced by physostigmine (100 nM) and the muscarinic receptor agonist, muscarone (10 nM–1 μM), and enhanced by atropine (0.1–100 nM). These findings indicate the presence of a muscarinic negative feedback mechanism controlling ACh release.The effects of various subtype-preferring muscarinic receptor antagonists were evaluated on [3H]-ACh release and muscle contraction. The rank potency (−log EC50) orders at pre- and postjunctional level were: atropine 4-diphenyl-acetoxy-N-piperidine (4-DAMP)>mamba toxin 3 (MT-3)>tripitramine>para-fluorohexahydrosiladiphenidol (pF-HHSiD)methoctraminepirenzepine>tripinamide, and atropine4-DAMP>pF-HHSiD>>pirenzepine=tripitramine>tripinamide>methoctramine>>MT-3, respectively.The comparison of pre- and post-junctional potencies and the relationship analysis with the affinity constants at human cloned muscarinic receptor subtypes indicates that the muscarinic autoreceptor inhibiting ACh release in human detrusor is an M4 receptor, while the receptor involved in muscular contraction belongs to the M3 subtype.Experiments were carried out in human detrusor strips to characterize muscarinic receptor subtypes involved in the prejunctional regulation of acetylcholine (ACh) release from cholinergic nerve terminals, and in the postjunctional smooth muscle contractile response.In detrusor strips preincubated with [3H]-choline, electrical field stimulation (600 pulses) delivered in six trains at 10 Hz produced a tritium outflow and a contractile response. In the presence of 10 μM paraoxon (to prevent ACh degradation) the tritium outflow was characterized by HPLC analysis as [3H]-ACh (76%) and [3H]-choline (24%).Electrically-evoked [3H]-ACh release was abolished by tetrodotoxin (TTX: 300 nM) and unaffected by hexamethonium (10 μM), indicating a postganglionic event. It was reduced by physostigmine (100 nM) and the muscarinic receptor agonist, muscarone (10 nM–1 μM), and enhanced by atropine (0.1–100 nM). These findings indicate the presence of a muscarinic negative feedback mechanism controlling ACh release.The effects of various subtype-preferring muscarinic receptor antagonists were evaluated on [3H]-ACh release and muscle contraction. The rank potency (−log EC50) orders at pre- and postjunctional level were: atropine 4-diphenyl-acetoxy-N-piperidine (4-DAMP)>mamba toxin 3 (MT-3)>tripitramine>para-fluorohexahydrosiladiphenidol (pF-HHSiD)methoctraminepirenzepine>tripinamide, and atropine4-DAMP>pF-HHSiD>>pirenzepine=tripitramine>tripinamide>methoctramine>>MT-3, respectively.The comparison of pre- and post-junctional potencies and the relationship analysis with the affinity constants at human cloned muscarinic receptor subtypes indicates that the muscarinic autoreceptor inhibiting ACh release in human detrusor is an M4 receptor, while the receptor involved in muscular contraction belongs to the M3 subtype.British Journal of Pharmacology (2000) 129, 493–500; doi:10.1038/sj.bjp.0703080
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