Yasuo Shimo

Heterogeneity of muscarinic receptors in the guinea pig esophageal muscularis mucosae and ileal longitudinal muscle

Pharmacologic characteristics of muscarinic receptors in the muscularis mucosae of the guinea pig esophagus were examined in vitro and compared with those of the longitudinal muscle of the guinea pig ileum. All cholinomimetics tested produced a sustained contraction of the muscularis mucosae, in a concentration-dependent manner, which was associated with a small biphasic change in membrane potential, initially a hyperpolarization followed by a depolarization. The contractile response was hardly modified by verapamil, but was depressed by calcium removal from the bathing medium. Both atropine and pirenzepine antagonized the contractile response in a competitive manner, with higher pA2 values than those in the ileum. These results indicate that the muscarinic receptors of the muscularis mucosae of the guinea pig esophagus mainly link with calcium ion channels which are independent of changes in membrane potential and that their subtype populations are probably pharmacologically distinct from those in the ileal longitudinal muscle.

Pharmacological differences of non‐adrenergic inhibitory response and of ATP‐induced relaxation in guinea‐pig tracheal strip‐chains

The presence of non-adrenergic inhibitory neurons in the guinea-pig tracheal muscle has been demonstrated by Coburn & Tomita (1973) and Richardson & Bouchard (1975), using isolated tracheal strips under isometric conditions. On the other hand, Coleman & Levy (1974) also recognized these neurons using the isolated tracheal tube preparation under isotonic conditions and proposed adenosine 5’-triphosphate (ATP) as a transmitter in these neurons. Previously (Kamikawa & Shimo, 1976), we reported that the inhibitory response to ATP of guinea-pig tracheal strip-chains is antagonized by indomethacin, aspirin and polyphloretin phosphate (PPP) so that this response may be an indirect one mediated by prostaglandin (PG) &. The present report thus describes the influences of these drugs on the nonadrenergic inhibitory response to field stimulation of tracheal strip-chains of guinea-pigs under isometric conditions. Male guinea-pigs, 300 to 6OOg, were used. The tracheal strip-chain was prepared according to the method described in our previous paper and suspended in a 30ml organ bath with modified Krebs-Ringer solution (composition mM: NaCI, 120; KCl, 4.7; CaClp, 2.5; MgCI2, 1-2; NaHCO,, 25; KH2POo, 1.2; glucose, 14; pH 7.4) aerated with 5 % C 0 2 in oxygen at 37 ’. The preparation was mounted under 0.5g of initial tension and contracted by an application of histamine 10 p~ before every stimulation. The tracheal response was recorded isometrically using a forcedisplacement transducer coupled to a Recticorder (Nihon Kohden). The field stimulation was with rectangular pulses of varied frequencies from 1 to 50Hz, pulse duration of 0.3 ms and supramaximal voltage, through bipolar platinum electrodes which were 10 mm apart and connected to a Nihon Kohden stimulator, model SEN 1101. The total number of stimulating pulses was kept constant at 60. For the elimination of parasympathetic components in responses to field stimulation, the Krebs-Ringer solution contained 1 p~ atropine sulphate. Drugs used were: adenosine 5’-triphosphate disodium salt (Sigma), histamine dihydrochloride, atropine sulphate (Wako Pure Chem.), guanethidine sulphate (Ciba-Geigy), propranolol hydrochloride, tetrodotoxin, indomethacin (Sankyo), and PPP (AB Leo). Indomethacin was dissolved in 50% ethanol, and all other drugs were dissolved in physiological saline. Field stimulation of the tracheal strip-chains with the stimulus parameters described above elicited only relaxation which depended on the stimulus frequency.

Inhibitory effects of catecholamines on cholinergically and non‐cholinergically mediated contractions of guinea‐pig isolated bronchial muscle

Abstract— The actions of catecholamines on the responses evoked by electrical field stimulation or by acetylcholine and substance P in guinea‐pig bronchial strip chain have been examined. Electrical field stimulation evoked a biphasic contraction, consisting of a cholinergically‐mediated fast contraction followed by a non‐cholinergically‐mediated slow contraction. All catecholamines tested caused a concentration‐dependent reduction in the height of the biphasic contraction, where non‐cholinergic contractions were more potently inhibited. The inhibitory effect of isoprenaline was largely prevented by propranolol (2 μM) alone, whereas those of noradrenaline and adrenaline were prevented by treatment with both propranolol (2 μM) and yohimbine (2 μM). The inhibitory effect of dopamine was unaffected either by propranolol (2 μM), yohimbine (2 μM) or haloperidol (10 μM). Submaximal contractions of bronchial muscle evoked by exogenous acetylcholine (2 μM) or substance P (0.2 μM) were also inhibited by catecholamines, except dopamine, but the effects were antagonized by propranolol (2 μM) alone. The results suggest that in guinea‐pig isolated bronchial muscle, catecholamines can inhibit both cholinergic and non‐cholinergic excitatory neurotransmissions not only by postjunctional β‐adrenoceptors but also by prejunctional α2‐adrenoceptors.

Cooling-induced supersensitivity to acetylcholine in the isolated airway smooth muscle of the rat

SummaryIsolated tracheal and bronchial strip-chain preparations of the rat were used to study the effect of temperature on electrically or acetylcholine-induced contraction. The preparations were suspended in the organ bath containing Krebs bicarbonate solution for isometric tension recording. A decrease of bath temperature from 37°C to 20°C (cooling) had no effect on basal tone but augmented the contractile responses of the trachea and bronchus caused by stimulation of intramural cholinergic nerves (0.5–5 Hz) or acetylcholine (3 μmol/1–0.3 mmol/l). Cooling-induced augmentation of the contractile response to acetylcholine was not affected by pretreatment of the tissue with physostigmine (0.1 μmol/l) or tetrodotoxin (0.3 μmol/l). The affinity of acetylcholine for the tracheal muscarinic receptors at 20°C, determined from its dissociation constant (KA), was not significantly different from that at 37°C. On the other hand, acetylcholine-induced contraction of trachea which was incubated with isosmotic K+-rich Krebs solution and with Ca-free, EGTA (0.1 mmol/l) containing Krebs solution were both augmented at 20°C. Caffeine or vanadate, each at a lower concentration than the threshold for causing contraction by itself, augmented the contractile responses of the trachea to acetylcholine (1 μmol/l–0.3 mmol/l). These potentiating effects of caffeine and vanadate were greater at 20°C then 37°C. From these observations, it is concluded that increased responsiveness of the rat airway smooth muscle to acetylcholine with lowered temperature may involve the acceleration of Ca release from intracellular storage sites, inhibition of Ca extrusion from the cell and or the inhibition of Ca reuptake by intracellular storage sites.

Mediation of prostaglandin E2 in the triphasic response to ATP of the isolated tracheal muscle of guinea-pigs

ATP, at a dose higher than 0·1 μg ml−1, showed a biphasic action consisting of an initial increase followed by a gradual decrease of muscle tension in the isolated tracheal strip‐chains of guinea‐pigs. The pattern of this biphasic response to ATP varied with the level of basal tone of the preparation at the moment of application of ATP. A similar biphasic action was obtained by prostaglandin (PG) E2 among the various active substances studied including acetylcholine, histamine, catecholamines and various types of PG. Indomethacin (0·1 μg ml−1) and aspirin (30 μg ml−1) completely abolished the ATP‐induced inhibitory response observed in the presence of histamine (10 μm). Polyphloretin phosphate (100 μg ml−1) also significantly depressed the inhibitory response to ATP or PGE2. It is concluded that the response to ATP of the preparation is mediated by PGE2 released via the stimulation of its biosynthesis.

Pharmacological characterization of the opioid receptor in the submucous plexus of the guinea-pig oesophagus

1 The cholinergically mediated electrically‐induced contractions of the submucous plexus‐longitudinal muscularis mucosae preparation of the guinea‐pig oesophagus were used to study the actions of opioid peptides and morphine. 2 The twitch contractions of the tissue (0.1 Hz, 0.5 ms, supramaximal voltage) were inhibited by all the opioid peptides and morphine in a concentration‐dependent manner. The order of potency was dynorphin‐(1–13) > α‐neo‐endorphin > β‐endorphin > [d‐Ala2]‐methionine‐enkephalin ≪≪ α‐endorphin, methionine‐enkephalin, leucine‐enkephalin and morphine. 3 The inhibitory actions of dynorphin‐(1–13) (20 nm), α‐neo‐endorphin (100 nm) and β‐endorphin (3 μm) were completely reversed either by naloxone (1 μm) or by morphine (100 μm). The Ke values of naloxone against dynorphin‐(1–13) and α‐neo‐endorphin were 30 and 25 nm, respectively. 4 Increasing the concentration of calcium from 1.8 to 3.6 mm in Tyrode solution decreased the sensitivity of the tissue to dynorphin‐(1–13) 7.4 times and to α‐neo‐endorphin 462 times. 5 The inhibitory actions of dynorphin‐(1–13) (100 nm) and α‐neo‐endorphin (300 nm) were inversely related to stimulus frequency, being most active at low frequencies (0.1–1 Hz), and least active at high (30 Hz). 6 Exogenously applied acetylcholine produced concentration‐dependent contractions of the isolated muscularis mucosae, with an EC50 of 72.6 ± 4.5 nm. The contractile response of the oesophagus to acetylcholine was not affected by the pretreatment of the tissue with dynorphin‐(1–13) (100 nm), α‐neo‐endorphin (300 nm) or β‐endorphin (3 μm). 7 It is concluded that the submucous plexus‐longitudinal muscularis mucosae of the guinea‐pig oesophagus is inhibited by opioid peptides acting at prejunctional opioid receptors, probably of the κ‐subtype.

Investigation into the 5-hydroxytryptamine-induced relaxation of the circular smooth muscle of guinea-pig stomach fundus

The 5-HT receptor that mediates relaxation of circular muscle strips of the guinea-pig stomach fundus under resting tone was investigated. Concentration-dependent relaxation was obtained in the presence of atropine (0.2 microM) with 5-hydroxytryptamine (5-HT) (apparent mean pEC50 value, 5.27), 5-carboxamidotryptamine (7.35), 5-methoxytryptamine (4.98) and 5-methyltryptamine (4.58). 1-(m-Trifluoromethyl-phenyl)piperazine and 8-hydroxy-2-(di-n- propylamino)tetralin acted as partial agonists while 2-methyl-5-hydroxytryptamine, alpha-methyl-5-hydroxytryptamine, sumatriptan, metoclopramide and cisapride had little or no effect on the guinea-pig stomach fundus. The concentration-response curve for 5-HT was not affected by tetrodotoxin (0.3 microM), guanethidine (5 microM) or indomethacin (2 microM), suggesting that the relaxation is non-neuronal in origin and is independent of the release of catecholamines or prostanoids. The non-selective 5-HT receptor antagonist, metitepine (0.03-0.1 microM), the 5-HT1C/5-HT2 receptor antagonists, mianserin (0.3-1 microM), pizotifen (0.3-1 microM), ketanserin (3-10 microM), and the 5-HT1A/5-HT2 receptor antagonist, spiperone (3 microM), shifted the concentration-response curves for 5-HT to the right. A 5-HT3 receptor antagonist, ICS205-930 (1 microM), propranolol (1 microM) and phentolamine (1 microM) failed to block the 5-HT-induced relaxation. In conclusion, the results found with agonists and antagonists are compatible with the view that a 5-HT1-like receptor is involved in 5-HT-induced direct relaxation of circular muscle of guinea-pig stomach fundus.

Investigation of nicotine-induced relaxation of circular smooth muscle of the guinea-pig gastric fundus

A possible mechanism for the nicotine-induced relaxation of circular muscle strips of the guinea-pig gastric fundus was investigated. In the presence of atropine (0.2 microM), nicotine produced concentration-dependent relaxation with a maximum effect at 100 microM (mean pEC50 value, 4.60). The maximum relaxation due to nicotine was greatly reduced by pretreatment with tetrodotoxin (0.3 microM) or hexamethonium (10 microM), but not with metitepine (0.3 microM). Combined pretreatment with timolol (0.3 microM) and phentolamine (0.3 microM) or chemical sympathectomy by 6-hydroxydopamine pretreatment partially inhibited the nicotine-induced relaxation. alpha-Chymotrypsin (2 u/ml) which abolished the equivalent relaxation induced by vasoactive intestinal polypeptide (VIP) had no effect on nicotine-induced relaxation. NG-Nitro-L-arginine (L-NNA) and NG-Nitro-L-arginine methyl ester (L-NAME) caused a concentration-dependent inhibition of the nicotine-induced relaxation (98% inhibition at 10 microM of L-NNA), but had no effect on sodium nitroprusside- or noradrenaline-induced relaxation. The inhibitory effect of L-NNA or L-NAME was reversed completely by L-arginine (3 mM), but not by D-arginine (3 mM). From these results, we concluded that nicotine-induced relaxation of the guinea-pig gastric fundus is mediated largely by the release of nitric oxide or a related substance and partially by the release of noradrenaline. Possible contributions of 5-hydroxytryptamine or VIP to the nicotine-induced relaxation appear to be negligible.

Cooling‐induced augmentation of the contractile response of the golden hamster tracheal muscle to substance P in‐vitro

The effect of temperature on the substance P‐induced contraction of the isolated tracheal strip‐chain preparation from the golden hamster has been examined. A decrease of bath temperature from 37 to 20°C augmented the contractile response of the trachea caused by substance P. Similar cooling‐induced augmentation was observed in the contractile responses to caffeine and carbachol, but not to fratassium chloride. The increased responsiveness with lowered temperature of the trachea to substance P may be due to the acceleration of Ca2+ release from an intracellular storage site.

Indirect action of 5‐hydroxytryptamine on the isolated muscularis mucosae of the guinea‐pig oesophagus

1 The site of action of 5‐hydroxytryptamine (5‐HT) was examined on the isolated muscularis mucosae attached to the submucous plexus of the guinea‐pig oesophagus. Isotonic responses of the longitudinal muscularis mucosae were recorded. 2 5‐HT produced a transient contraction of the muscularis mucosae at concentrations higher than 3 μm. The contraction was rapid in onset, reaching a peak in about 15 s or less, and was restored to the basal level after 20 to 30 s without washing out 5‐HT. When the 5‐HT‐induced contraction faded to the basal tone, successive applications of 5‐HT no longer produced any contracture. 3 Nicotine (Nic), at concentrations higher than 10 μm, also produced a transient contraction which had a very similar pattern to that induced by 5‐HT. Again, the successive application of Nic no longer produced any contracture following prior treatment with Nic itself. However, the 5‐HT‐induced contraction was not modified by the presence of Nic. 4 Exogenously applied acetylcholine (ACh) produced a concentration‐dependent contraction of the muscularis mucosae, the 50% effective concentration (EC50) was 69 ± 5.6 nm. The contraction was sustained during incubation with ACh, and was not modified by prior treatment with 5‐HT or Nic. 5 The 5‐HT (100 μm)‐induced contraction was completely abolished by tetrodotoxin (0.2 μm) and atropine (0.2 μm). This means that the action is mediated by stimulating cholinergic nerves in the submucous plexus attached to muscularis mucosae. Moreover, the stimulating action of 5‐HT does not involve nicotinic receptors, since the action was not blocked by hexamethonium (100 μm). 6 Among several 5‐MT antagonists examined, methysergide (1 μm), ketanserin (1 μm) and morphine (100 μm) failed to modify the 5‐HT (100 μm)‐induced contraction significantly. Cinanserin (0.1–3 μm), cyproheptadine (3–100 nm) and phenoxybenzamine (0.1–3 μm) inhibited the 5‐HT‐induced contraction, in a concentration‐dependent manner, and each highest concentration abolished the response. However, none of these antagonists was specific for 5‐HT, but the Nic (100 μm) or ACh (0.1 μm)‐induced contractions were also inhibited by them. 7 The present results indicate that 5‐HT contracts the muscularis mucosae of the guinea‐pig oesophagus indirectly by stimulating cholinergic nerves in the submucous plexus, and has no direct action on the muscularis mucosae. In addition, the type of 5‐HT receptors responsible for the stimulant action may be different from those in other parts of the gastrointestinal tract, blood vessels or brain, because of the different effects of 5‐HT antagonists.