The effects of metoclopramide on acetylcholine release and on smooth muscle response in the isolated guinea-pig ileum

Summary The effects of metoclopramide on smooth muscle contraction and on release of acetylcholine were studied in the guinea-pig myenteric plexus longitudinal muscle preparation. Acetylcholine was determined either as endogenous acetylcholine, or as labelled transmitter from strips preloaded with ³H-choline.Metoclopramide caused an increase in resting tension of longitudinal muscle as well as an increase in resting output of either endogenous or labelled acetylcholine. Tetrodotoxin abolished the metoclopramide-evoked increase in transmitter release. The increase in smooth muscle tension was clearly related to the increase in resting output. The effects of metoclopramide on both longitudinal muscle contraction and resting release of labelled acetylcholine were prevented in the presence of a concentration of 5-hydroxytryptamine (5-HT) that desensitized 5-HT receptors. This suggests that metoclopramide stimulates neuronal 5-HT receptors and, thereby, facilitates acetylcholine release.Metoclopramide augmented the twitch-like contractions induced by field stimulation at 0.1 Hz. Contractions elicited at 1 Hz were only slightly enhanced. Similarly, metoclopramide facilitated only the release of labelled acetylcholine evoked by electrical stimulation at 0.1 Hz, but not that at 1 Hz. The facilitatory effetts of metoclopramide on twitch height and evoked release could not be attributed to a blockade of presynaptic inhibitory -adrenoceptors, dopamine or muscarine receptors.     

Fading and tachyphylaxis to the contractile effects of substance P in the guinea-pig ileum

Substance P (SP) caused an immediate and vigorous contraction of the longitudinal smooth muscle layer of the guinea-pig ileum. The contractile response to SP, unlike that to acetylcholine or histamine was not maintained but faded to baseline levels in about 6 min. When 0.3-1.0 nM SP was added the fading time was shorter than 6 min and tachyphylaxis did not develop. Higher concentrations of SP produced fading times of about 6 min that could not be increased even by adding extremely high concentrations of the peptide, up to 1800 nM. Short fading times and the lack of development of tachyphylaxis are the result of the rapid adsorption and/or metabolism of SP. The addition of exogenous peptidases such as pronase, chymotrypsin and an extract of black widow spider venom gland dramatically increased the rate of degradation of SP, shortened the fading response and blocked the development of tachyphylaxis. Tetrodotoxin and atropine reduced the fading time by 25%, while eserine increased its duration several-fold; these findings are consistent with the existence of a cholinergic nerve component in the mediation of some of the effects of SP receptor and, in part, to adsorption and metabolism of the peptide. The magnitude of the tachyphylaxis to SP was proportional to the concentration of the desensitizing dose of the peptide and was specific to SP and to the related peptide physalaemin; no cross-tachyphylaxis towards other agents was found.     

The effect of substance P on nerve action potential propagation and cholinergic transmission in the myenteric plexus of the guinea-pig ileum

Summary The effect of substance P on nerve terminals in myenteric plexus of the guinea-pig ileum was investigated. Neurogenic twitches of the myenteric plexus longitudinal muscle strip were recorded. Twitches of the strip portion where excitation involved the most distal parts of cholinergic nerve terminals were more increased by local application of substance P (0.1 and 0.4 nmol/l) than twitches of the portion where excitation involved both distal and proximal parts of nerve terminals. Substance P addition to a portion of the strip conducting nerve action potentials to invade the neighbouring strip portion also augmented twitches of the latter portion so that the interference with the propagation process was considered. The effect of substance P was poorly antagonized by the addition of a substance P antagonist, (D-Arg¹, D-Pro², D-Trp^7,9, Len¹¹)-substance P. Compound nerve action potentials were evoked in strands of fibres of the myenteric plexus by low-frequency train stimulation (1 Hz). The addition of substance P prevented a decrease of the amplitude of responses observed under control conditions. Using high-frequency train stimulation (30 Hz) the amplitude of responses to impulses 2-7 was augmented over that to the first impulse; substance P further increased such facilitation regularly. It seems that substance P might promote nerve action potential invasion of the distal parts of nerve terminals. Send offprint requests to O. Kadlec at the above address     

Electrophysiological and mechanical characteristics of histamine receptors in smooth muscle cells of the guinea-pig ileum

To elucidate the role of the histamine receptor in functions related to intestinal motility, we investigated the effects of histamine and its antagonists on electrical and mechanical activities in longitudinal and circular layers of the terminal region of the guinea-pig ileum. Histamine hyperpolarized the membrane in the circular smooth muscle cells by increasing the permeability of K+ and it transiently inhibited generation of the spike while resting tone was elevated. Cimetidine (CIM) inhibited the hyperpolarization and relaxation induced by histamine while mepyramine (MEP) inhibited the contraction but did not affect the histamine-induced hyperpolarization. In the presence of CIM, histamine did not depolarize the membrane but did lower the threshold potential required for generation of the spike potential, increased the appearance of the spike and enhanced the phasic contraction. Histamine, in 20 mM K+ solution, hyperpolarized the membrane and produced a biphasic response, an initial relaxation and a subsequently generated contraction, in a concentration-dependent manner. In the longitudinal smooth muscle cells, histamine depolarized the membrane, and enhanced both generation of the spike and the contraction. MEP (0.1 microM) but not CIM (1 microM) blocked the histamine-induced responses. CIM at a higher concentration (10 microM) enhanced the histamine-induced contraction, while histamine did not relax the tissue precontracted by 20 mM K+. These results indicate that the circular muscle cells have both H1 and H2 receptors while the longitudinal muscle cells have the H1 receptor. The excitatory responses induced by activation of the H1 receptor in smooth muscle cells differ in these layers.     

Post-tetanic potentiation in the innervated smooth muscle preparation of the guinea-pig ileum

The phenomenon of posttetanic potentiation of electrically evoked twitch contractions in the myenteric plexus-longitudinal muscle of the guinea-pig ileum was observed when (1) post-tetanic inhibition had been prevented by naloxone, an opiate receptor antagonist, and (2) the twitches had been diminished by indomethacin, an inhibitor of prostaglandin synthesis. The origin of posttetanic potentiation at this muscarinic synapse appeared to be presynaptic.     

Actions of quinidine and apamin on after-hyperpolarization of the spike in circular smooth muscle cells of the guinea-pig ileum

Summary The effects of quinine and quinidine on membrane potential and action potential were investigated in circular smooth muscle of the guinea-pig ileum and the findings compared with the actions of apamin. In addition to results obtained from microelectrode experiments, the actions of quinidine and apamin on membrane currents were assessed using the single cell voltage clamp method. Quinine (above 0.2 mmol/l) and quinidine (above 0.08 mmol/l) depolarized the membrane, increased the membrane resistance and blocked generation of the after-hyperpolarization of the spike. Higher concentrations of both agents reduced the amplitude of the action potential and further depolarized the membrane. Quinidine and quinine possessed much the same action, with the former being more potent than the latter. Apamin, an inhibitor of the Ca-dependent K current, did not inhibit the after-hyperpolarization of the spike and had no effect on the membrane potential. In voltage clamp experiments, a depolarizing pulse (above –30 mV from –60 mV; 200 ms duration) elicited an inward current, followed by an outward current. With application of 2.5 mmol/l Mn instead of Ca, the outward current was subclassified into the Mn sensitive (Ca-dependent) and Mn resistant (volage-dependent) K currents. Apamin (0.1 mol/l) did not modify membrane currents evoked in the circular muscle cell, while, 0.1 mmol/l quinidine inhibited both the Ca- and voltage-dependent K outward currents, and Ca inward current.Our observations suggest that apamin-insensitive Ca-dependent K channels are present in the smooth muscle membrane and that they probably participate in the falling phase and after-hyperpolarization of the action potential.     

Impulse transmission in the myenteric plexuslongitudinal muscle preparation of the guinea-pig ileum

1. In a preparation consisting of the myenteric plexus and the longitudinal muscle layer removed from a segment of guinea-pig ileum, spontaneous action potentials occurred which were unaffected by tetrodotoxin but suppressed by Mn(2+) and were therefore myogenic.2. A single current pulse of 0.1 ms duration evoked a response consisting of an early action potential followed after a delay of about 200 ms by a complex of biphasic spikes. The first action potential was conducted for no more than 15 mm and the second complex for 30-70 mm.3. Since the first action potential was unaffected by hyoscine or Mn(2+) but abolished by tetrodotoxin, it was due to excitation of nerve fibres. The later complex of spikes was suppressed by hyoscine and Mn(2+) and therefore due to excitation of smooth muscle. It was also inhibited by adrenaline or morphine, compounds which depress acetylcholine release. The evoked smooth muscle response was followed by absence of spontaneous electrical activity for 2-4 seconds.4. The nerves travelling in a longitudinal direction had a mean maximum conduction velocity of 0.65 m/s, an absolute refractory period of 2.8 ms and a relative refractory period of about 20 ms.5. The conduction velocity of the smooth muscle response evoked by stimulation of the nerve with a single pulse was 0.16 m/second. After a single pulse the muscle was inexcitable for 0.7-1.3 s; the delay of transmission from nerve to muscle was 210 ms. When instead of a single pulse a train of two-five pulses at 20 ms intervals was applied, the size, conduction distance and conduction velocity of the evoked smooth muscle response were increased.     

Effects of Gymnodinium breve toxin on the smooth muscle preparation of guinea-pig ileum

1 The effects of Gymnodinium breve neurotoxin (GT) on smooth muscles were studied using the guinea-pig isolated ileum.2 The toxin caused strong spasmogenic effects at 1-4 mug/ml, characterized by prolonged tonic contraction with superimposed pronounced pendular movements. Tachyphylaxis was observed upon administration of successive doses.3 Atropine blocked the contractile response elicited by GT, whereas mepyramine and hexamethonium failed to do so. These findings tentatively suggested a cholinergic involvement at a post-ganglionic site of action.4 In the presence of tetrodotoxin the effects of GT were abolished, excluding direct action of the toxin on the smooth muscle.5 It is concluded that GT exerts its spasmogenic effects through stimulation of the post-ganglionic cholinergic nerve fibres.     

Effects of gymnodinium breve toxin on the smooth muscle preparation of guinea-pig ileum

1 The effects of Gymnodinium breve neurotoxin (GT) on smooth muscles were studied using the guinea-pig isolated ileum.2 The toxin caused strong spasmogenic effects at 1-4 mug/ml, characterized by prolonged tonic contraction with superimposed pronounced pendular movements. Tachyphylaxis was observed upon administration of successive doses.3 Atropine blocked the contractile response elicited by GT, whereas mepyramine and hexamethonium failed to do so. These findings tentatively suggested a cholinergic involvement at a post-ganglionic site of action.4 In the presence of tetrodotoxin the effects of GT were abolished, excluding direct action of the toxin on the smooth muscle.5 It is concluded that GT exerts its spasmogenic effects through stimulation of the post-ganglionic cholinergic nerve fibres.     

Photodynamic effects of erythrosine on the smooth muscle cells of guinea-pig taenia coli

Photon activation of the halogenated fluorescein derivative erythrosine caused a marked calcium-dependent contraction of the smooth muscle cells of the guinea-pig taenia coli superfused in vitro. Neither high intensity illumination alone (up to 5 X 10(4) lux) nor erythrosine alone (up to 2 X 10(-4) M) altered the tone of the taenia or its ability to respond to carbachol (5 X 10(-5) M); photo-irradiation of erythrosine before tissue contact was also ineffective. The magnitude of the photodynamic contraction was dependent upon the concentration of erythrosine, the intensity and wavelength of the incident light, and the presence of oxygen; indirect effects via neurotransmitter release or cyclo-oxygenase activation were specifically excluded. The photodynamic response was blocked by zero-[Ca]o and addition of EGTA (1 mM) but not by omission of [Mg]o or a decrease in [Cl]o or [Na]o. D600 (methoxyverapamil) 10(-5) M, or a ten fold increase in [Mg]o, to 11.3 mM, partly inhibited the photodynamic contraction at low, but not high, light intensities. These observations are consistent with the following sequence of events: (i) photo-activation of the erythrosine molecule, (ii) the generation of highly reactive singlet oxygen, (iii) local peroxidation of cell membrane proteolipid, (iv) increased membrane permeability to Ca2+, (v) the influx of Ca2+ and, (vi) muscle contraction. It is concluded that the photodynamic action of erythrosine presents a novel method for modulation of membrane calcium permeability, and hence [Ca]i, not only in smooth muscle but possibly in other cells as well, e.g., secretory, epithelial and myocardial cells.     

Protein kinase C regulates smooth muscle tension in guinea-pig trachea and ileum

To explore the function of protein kinase C in smooth muscle, the effects of phorbol esters, potent activators of protein kinase C, were examined in guinea-pig tracheal rings and ileal strips. In tracheal rings, phorbol-12,13-diacetate (PDA) and 12-deoxyphorbol-13-isobutyrate (DPB), both potent stimulants of protein kinase C, produce a concentration dependent, reversible relaxation of resting tracheal tension, whereas phorbol, an inactive analogue is ineffective. PDA also reverses contractions produced by carbachol, serotonin, prostaglandin F2 alpha and prostaglandin D2. In contrast to their ability to inhibit agonist induced contractions, PDA and DPB greatly amplify the constriction produced by a depolarizing concentration of KCl (59 mM). The calcium channel blockers verapamil, nifedipine and diltiazem block the constriction produced by KCl and PDA suggesting that under depolarizing conditions, PDA synergizes with increased intracellular calcium to potentiate muscle contraction. Similar biphasic responses to phorbol esters are elicited in strips of guinea-pig ileum. These results indicate that in addition to enhancing the actions of intracellular calcium in producing contraction, protein kinase C can also activate feedback mechanisms which limit cellular responses.     

Some effects of orthovanadate on smooth muscle cells of guinea-pig taenia caeci

The effect of orthovanadate on the response caused by the beta-adrenergic receptor agonist isoprenaline and on the ATP response were investigated by measuring potential changes and changes in the contractile state of smooth muscle cells of guinea-pig taenia caeci at 35 degrees C using the sucrose-gap method. The contraction evoked by carbachol (10(-7) M) or potassium (15 mM) was not modified by orthovanadate (1.0 mM). The hyperpolarization evoked in smooth muscle cells by ATP (0.4 mM) was affected to some extent by orthovanadate (1.0 mM), but was not modified in the absence of extracellular calcium. The hyperpolarization caused by isoprenaline (3 X 10(-6) M), however, was inhibited completely both in the presence and absence of calcium. These results are consistent with the view that isoprenaline activates calcium extrusion from the smooth muscle cells, a process being electrogenic in nature.     

Histamine-induced inositol phospholipid breakdown in the longitudinal smooth muscle of guinea-pig ileum.

The characteristics of histamine-stimulated inositol phospholipid breakdown in slices of guinea-pig ileal smooth muscle and cerebellum have been investigated. In cerebellar slices the inhibition of the inositol phospholipid response to histamine by mepyramine was consistent with competitive antagonism of histamine H1-receptors. In slices of the longitudinal smooth muscle of guinea-pig ileum, mepyramine produced only a weak inhibition of the response to histamine, at concentrations up to 1 microM. This was in striking contrast to the potent competitive antagonism of the H1-mediated contractile responses obtained with mepyramine in this tissue. The H1-receptor antagonists (+)-chlorpheniramine and promethazine similarly had no effect on the EC50 value for histamine in guinea-pig ileum, while promethazine competitively antagonized the muscarinic receptor-mediated inositol phospholipid response in this tissue (Ka 3.6 X 10(7)M-1). Cimetidine, on its own, did not significantly inhibit the inositol phosphate accumulation elicited by histamine in ileum. In the presence of 0.2 microM mepyramine, cimetidine (0.1 mM) produced a small parallel shift of the histamine concentration-response curve (Ka 3 X 10(4) M-1). This inhibition, however, was not consistent with antagonism of an H2-receptor-mediated response. The effect of a range of histamine analogues on inositol phospholipid breakdown was determined. Dose-response curves were constructed and characterized in terms of the EC50, slope and maximal response attainable relative to histamine. The H1-agonists, N alpha,N alpha-dimethylhistamine, N alpha-methylhistamine, 2-pyridylethylamine and 2-thiazolyethylamine produced the largest accumulations of [3H]-inositol-1-phosphate. A very weak response was produced by the H2-selective agonist impromidine, while dimaprit (also H2-selective) was without significant effect. Mepyramine appeared to antagonize competitively the response to the H1-selective agonist 2-pyridylethylamine. This was in contrast to the data obtained with other H1-agonists, where mepyramine produced only a small dextral shift of the agonist curves at low agonist concentrations and an increase in the Hill coefficient. This was particularly striking in the case of 2-methylhistamine. The results suggest that an H1-receptor component in guinea-pig ileum, may coexist with a larger inositol phospholipid response to histamine which is independent of the activation of H1- or H2-receptors.     

Comparative study of the effects of 4-aminopyridine and tetraethylammonium on neuro-effector transmission in the guinea-pig vas deferens.

1 Effects of 4-aminopyridine (4-AP) and tetraethylammonium (TEA) on the neuro-effector junction of the guinea-pig vas deferens were investigated by microelectrode and double sucrose gap techniques. 2 4-AP (0.05 to 0.5 mM) or TEA (0.5 to 1 mM) did not alter the membrane potential, or the membrane input resistance of the smooth muscle cell. 3 The amplitude and frequency of the miniature junction potentials (m.e.j.ps) were not modified by treatment with 4-AP (0.05 to 0.5 mM) or TEA (1 mM). 4 4-AP (1 mM) increased the membrane input resistance, enhanced the spike amplitude of the smooth muscle cells and thereby augmented the amplitude of twitch contraction. 5 4-AP (.05 to 0.5 mM) or TEA (1 mM) markedly increased the amplitude of excitatory junction potentials (e.j.ps), but the facilitation phenomena produced by repetitive stimulation were not affected. 6 The duration of the extracellularly recorded action potential from the small nerve bundle was prolonged by 4-AP (0.5 mM). 7 The amplitude of the e.j.p. was dependent on the external concentration of calcium. A straight line was produced when the amplitude of the e.j.p. and [Ca]o was plotted on a double log scale. Application of 4-AP resulted in a parallel shift of this line to the left. 8 These results indicate that 4-AP (0.05 to 0.5 mM) and TEA (0.5 to 1.0 mM) prolonged the action potential generated from the sympathetic nerve terminal thus enhancing the amplitude of the e.j.p. due to an increase in the Ca-influx. However, in the concentrations used, these compounds did not modify the Ca-mobilization in the nerve terminal or the postsynaptic membrane during the resting state.     

The effects of LSD in the guinea-pig ileum

The effects of lysergic acid diethylamide (LSD) on acetylcholine release and on smooth muscle tone were studied in the myenteric plexus-longitudinal muscle preparation of the guinea pig. LSD (0.01-10 microM) depressed in a concentration-dependent manner the electrically-evoked [3H]-acetylcholine outflow from strips preincubated with [3H]-choline. The maximal effect was a 45% inhibition by 1 microM LSD. The spontaneous outflow was not affected. Metitepine competitively antagonized (pA2 8.0) the LSD-induced reduction of the evoked outflow. Tolazoline and mepyramine did not affect the inhibitory action of LSD. The contractions in response to electrical stimulation were enhanced by 34% in the presence of 0.1 microM LSD. Other concentrations of LSD did not affect the twitches. LSD caused an increase in muscle tone which was antagonized non-competitively by mepyramine, metitepine and ketanserin. Ketanserin was a competitive antagonist against the histamine-induced contractions of the longitudinal muscle (pA2 8.49). The results suggest that LSD stimulates presynaptically located 5-HT receptors and thereby decreases the evoked acetylcholine release. In addition, LSD increases smooth muscle tone either directly through stimulation of H1 receptors or indirectly via histamine release.