Removal of the epithelium potentiates acetylcholine in depolarizing canine bronchial smooth muscle

Experiments were designed to determine whether the airway epithelium affects the membrane potential of the underlying smooth muscle. The effect of epithelium removal (by gentle rubbing) on the responsiveness of isolated canine bronchi was studied. Simultaneous recordings of mechanical and electrical activity were made in paired circumferential strips (with and without epithelium) of third-order bronchi. Changes in tension were recorded with a force transducer, and changes in membrane potential were measured with a microelectrode. The cell membrane potential and resting tension of the bronchial smooth muscle were stable over a 150-min period and were not affected by removal of the epithelium. In the presence of antagonists at muscarinic and adrenergic receptors, the resting tension and membrane potential were comparable in preparations with and without epithelium. By contrast, the anticholinesterase, echothiophate, caused depolarization in bronchi without epithelium. Exposure to high potassium induced similar levels of depolarization and contraction in tissues with and without epithelium. No significant differences in threshold for depolarization or for mechanical activation in the membrane potential-tension relationship were noted in the presence or absence of epithelium. In the presence of echothiophate, removal of the epithelium augmented the contraction of the bronchi to acetylcholine; the depolarization of the cell membrane induced by the cholinergic transmitter was significantly larger than in control tissues, even when matched contractions were compared. These observations indicate that the respiratory epithelium generates an inhibitory substance that dampens depolarization and contraction of bronchial smooth muscle caused by acetylcholine.     

Neuromuscular modulation in Limulus by both octopamine and proctolin

Both octopamine and proctolin potentiate nerve-evoked skeletal muscle contractions in the horseshoe crab, Limulus. The threshold concentration for octopamine was 10^?9 to 10^?8M, while for proctolin it was 3 × 10^?9M. Norepinephrine and dopamine produced effects similar to octopamine but at higher thresholds; tyramine and serotonin were ineffective. Octopamine caused significant increases in amplitudes of excitatory postsynaptic potentials (epsps) of muscle fibers, but had little effect on muscle fiber input resistance or membrane potential. Also, octopamine did not affect depolarization of muscle fibers and subsequent contraction due to the direct action of exogenously applied glutamate. These results suggest that octopamine potentiates nerve-evoked contractions primarily by facilitating release of neuromuscular transmitter. At concentrations above 10^?7M, however, octopamine sometimes caused muscle spikes in response to motoneuron stimulation, a finding that suggests that octopamine may also have some postsynaptic action. Proctolin potentiated the muscle contractions evoked by glutamate but had little effect on glutamate-evoked muscle fiber depolarization, muscle fiber input resistance, or membrane potential. Thus, proctolin appears to act directly on skeletal muscle to enhance contractility. The proctolin-induced potentiations of contraction were sometimes accompanied by modest increases in epsp amplitude, so that unlike lobster skeletal and Limulus cardiac neuromuscular preparations, proctolin may have a secondary direct synaptic effect. Both octopamine and proctolin have been found in Limulus cardiac ganglion. This potential access to the hemolymph and the relatively low threshold concentrations needed for physiological action suggest that octopamine and proctolin could function as hormonal modulators of neuromuscular function in Limulus.     

4-Aminopyridine-induced changes in the electrical and contractile activities of the gastric smooth muscle

Effects of 4-aminopyridine (4-AP) on the electrical and contractile activities of the fundus and antrum of the cat stomach were studied using the sucrose-gap technique. In the fundus, low concentrations of 4-AP (up to 1 mmol/l) induced membrane depolarization and appearance of spike potentials and phasic contractions. After preliminary administration of atropine, 4-AP produced an opposite effect: hyperpolarization and relaxation. On the background of tetrodotoxin (TTX) plus antagonists of cholinergic and adrenergic receptors, high concentrations of 4-AP (greater than 5 mmol/l) caused membrane depolarization and appearance of spike potentials and phasic contractions. In the antrum, 4-AP in low concentrations (up to 1 mmol/l) decreased both the amplitude and the duration of the second component of the plateau-action potential, as well as those of the phasic contractions. This effect decreased in the presence of adrenergic receptor antagonists and was abolished by TTX. On this background, high concentrations of 4-AP (greater than 5 mmol/l) led to the appearance of spike potentials superimposed on the second component of the plateau-action potentials, and to a further increase in the phasic contraction amplitudes. The present data suggest that 4-AP exerts its effects via an increase in neurotransmitter release (low concentrations) and/or directly on the smooth muscle cell membrane (high concentrations).     

Postsynaptic alpha adrenoceptors on vascular smooth muscle

A heterogeneous population of alpha adrenoceptors mediates vasoconstriction in the canine saphenous vein (CSV). Studies with isolated strips of venous smooth muscle incubated with selective alpha-adrenoceptor agonists and antagonists revealed that both alpha 1 and alpha 2 adrenoceptors exist independently in this tissue and both subtypes mediate a contractile response. Measurement of contractile responses in reduced or zero external calcium conditions indicates that stimulation of alpha 1 adrenoceptors induces contractions by influx of extracellular calcium and release of calcium from internal stores. In contrast, 45Ca uptake studies suggest that activation of the postsynaptic alpha 2 adrenoceptor produces vasoconstriction dependent only on influx of extracellular calcium. The influx of calcium produced by the selective alpha 2-adrenoceptor agonist BHT-920 is inhibited by calcium entry blockers. Measurements of transmembrane potentials from smooth muscle cells of the CSV suggest that alpha 1-adrenoceptor activation produces depolarization and contraction (electromechanical coupling) whereas alpha 2-adrenoceptor stimulation does not result in concentration-dependent depolarization of the smooth muscle cells (pharmacomechanical coupling).     

Localization and function of metabotropic glutamate receptor 8 in the enteric nervous system

The enteric nervous system (ENS) contains glutamatergic neurons, transporters, and functional ionotropic and groups I and II metabotropic glutamate receptors (mGluRs). The aim of this study was to determine whether the ENS contains functional group III mGluRs. RT-PCR demonstrated the expression of mGluR7 and mGluR8 mRNA in rat myenteric ganglia. Western blot analysis confirmed the presence of mGluR8 protein. Immunocytochemistry, in conjunction with confocal microscopy, demonstrated mGluR8 immunoreactivity in the ENS of several species, including humans. mGluR8 immunoreactivity was localized to the membrane of nerve cell bodies that received glutamatergic input. Significant receptor internalization of mGluR8 was observed on activation, and localization to membrane was observed on blocking with the mGluR III antagonist (RS)-cyclopropyl-4-phosphonophenylglycine (CPPG). mGluR8-positive myenteric neurons contained glutamate or nitric oxide synthase (NOS), a marker of inhibitory motorneurons. Enteric group III mGluRs are functional because mGluR8 agonists inhibited forskolin-induced accumulation of cAMP in isolated myenteric ganglia, and CPPG reduced this effect. In addition, an accelerating effect on guinea pig colonic motility was observed after the application of mGluR8 agonists. Increase in motility was specific, because CPPG inhibited it. Moreover, in the presence of hexamethonium or Nomega-nitro-l-arginine methyl ester, an inhibitor of NOS, responses caused by mGluR8 agonists were abolished. mGluR8 agonists also increased longitudinal muscle contractions. These findings suggest that mGluR8 agonists increase motility by inhibiting nitrergic relaxation and possibly by facilitating cholinergic contractions.     

Effect of nicotine and acetylcholine on crustacean muscle membrane potential

We have investigated the effect of nicotine and acetylcholine on the resting membrane potential of the crayfish extensor muscle in order to determine whether crustacean muscle can be activated by cholinergic compounds. Intracellular recordings from individual deep extensor abdominal muscle cells were made using standard glass microelectrode techniques. The resting membrane potential was measured before and after treatment with glutamate, nicotine, and acetylcholine. Glutamate, which is a known activator of crayfish muscle, was used to determine whether the muscle cell preparation was viable and capable of responding to any of the test substances. Our results confirm that application of glutamate is associated with a depolarization of the muscle membrane. However, muscle cells showed no depolarization after treatment with nicotine (50 microM) or acetylcholine (66 microM). These results argue against the notion that increases in muscle tension may be responsible for the increased receptor organ discharge observed in the presence of nicotine. Rather, it supports the hypothesis that nicotine is acting directly on the mechanoreceptor membrane to change its sensitivity.     

Effects of cholinergic drugs on muscle contraction in Moniliformis moniliformis (Acanthocephala)

In whole Moniliformis moniliformis spontaneous muscle contractions were rhythmic; longitudinal contractions were measured with a force transducer. The cholinergic agonists levamisole and nicotine significantly increased muscle tension in whole worms; these contractions were tonic and were antagonised by the ganglionic blocker pentolinium and by piperazine. In addition, levamisole-induced contractions were inhibited by gallamine, hexamethonium, and norepinephrine. In worm segments, where drugs in solution were injected through the worms, acetylcholine (ACh) and nicotinic agonists were effective in causing contractions, whereas muscarinic agonists in concentrations up to 1 mM had no effect. Although muscle contraction in M. moniliformis was induced by nicotinic agonists, these contractions were effectively antagonised by a range of chemicals that block ganglionic, skeletal, and muscarinic sites in vertebrates. The presence of ACh in M. moniliformis and the effects of nicotinic agonists on muscle contraction suggest that ACh is a putative excitatory neurotransmitter.     

Depolarization of the Internal Membrane System in the Activation of Frog Skeletal Muscle

"Skinned" muscle fibers, single fibers from the frog semitendinosus muscle in which the sarcolemma had been removed, could be reversibly activated by electrical stimulation. Electrical responsiveness was abolished when the skinned fiber was prepared from a muscle exposed to a cardiac glycoside, and the development of responsiveness was delayed when the muscle was bathed in high potassium solution. The findings were taken as evidence that active sodium-potassium exchange across the internal membranes restored electrical excitability, after the sarcolemma had been removed, by establishing a potential gradient across the internal membranes. In general, the contractions were graded with the strength of the applied current. On occasion, however, "all-or-none" type responses were seen, raising the possibility that the internal membranes were capable of an electrically regenerative response. Activation could also be produced by an elevation of the intracellular chloride ion concentration or a decrease in the intracellular potassium, ion concentration, suggesting that depolarization of some element of the internal membrane system, that is, a decrease in the potential of the lumen of the internal membrane system relative to the potential of the myofibrillar space, was responsible for activation in these experiments. The distribution of both the electrically induced contractions and those produced by changes in the intracellular ion concentrations indicated that the responsive element of the internal membrane system was electrically continuous over many sarcomeres.     

Effect of manganese and verapamil on electrical and contractile responses of pulmonary artery smooth muscle induced by noradrenaline and histamine

Action of noradrenaline and histamine on the resting potential, membrane resistance and contractility of rabbit pulmonary artery muscle cells was investigated in normal and Ca-blockers (manganese and verapamil)-containing Ringer-Lock solutions. It was shown that catecholamine and histamine induced depolarization by different mechanisms. Thus, noradrenaline action is accounted for by the decreased membrane permeability to potassium ions, while the histamine-induced depolarization is a consequence of sodium and, probably, chlorine permeability. The contraction induced by the transmitters is activated primarily by the extracellular calcium ions entering the cells by two ways: via chemosensitive Ca-channels activated by adrenergic and histaminergic receptors or via potential-dependent slow Ca-channels activated by the transmitter-induced membrane depolarization. It is not excluded that during activation of muscle cells by the transmitters part of calcium is release from both intramembrane and intracellular stores.     

Cooperative interaction of glutamate and aspartate with receptors in the neuromuscular excitatory membrane in walking limbs of the lobster

When applied to lobster muscle fibers, L-glutamate, L-aspartate, and combinations of the two amino acids can induce membrane depolarization. Under normal conditions, a quantitative analysis of the depolarization response or change in membrane conductance was precluded by nonlinearities in the voltage—current relationship of the membrane. By including γ-aminobutyrate (GABA) in the bathing medium, the voltage—current relationship was made linear in the depolarizing direction over a range of 15–20 mV from the resting potential. However, a meaningful examination of the increase in membrane conductance caused by glutamate and aspartate was still not possible. Therefore, the depolarization responses caused by the excitatory amino acids were taken as a quantitative reflection of receptor activation in the excitatory postsynaptic membrane. In the presence of GABA, aspartate by itself, at concentrations up to 10 mM, had little excitatory activity, whereas glutamate effected an appreciable membrane depolarization at concentrations of 0.1 to 0.2 mM. Aspartate, at concentrations which exhibited no activity alone, markedly enhanced the excitatory action of glutamate. Aspartate shifted the glutamate dose-response curve to the left, but did not appear to affect the maximum depolarization response elicited by glutamate. These observations are consistent with the concept that aspartate increases the affinity between glutamate and the glutamate binding sites. Limiting slopes of log-dose versus log-response curves for the excitatory action of glutamate suggest that the interaction of glutamate with excitatory receptors is a cooperative process. The possibility exists that individual receptors contain multiple and distinct glutamate and aspartate binding sites. These results support the view that neuromuscular excitation in the lobster is mediated by glutamate and asparate functioning synergistically.     

Pharmacological properties of <Emphasis Type="SmallCaps">l</Emphasis>-glutamate receptors associated with the crayfish hindgut

Pharmacological agents were used to characterize glutamate receptors associated with crayfish hindgut. l-Glutamate reliably increased tonus in isolated hindguts of Procambarus clarkii and suppressed spontaneous hindgut contractions at concentrations of 10 micromol l(-1) or higher. Quisqualate and ibotenate mimicked the effects of L-glutamate. Experiments with strips and rings of hindgut tissue indicate that glutamate acts on both circular and longitudinal muscles. Hindgut contractions were not affected by (+/-)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionoic acid, N-methyl- d-aspartate, domoate or the metabotropic glutamate receptor agonist, (1S,3R)-1-amino-1-carboxycyclopentane-3-acetic acid. Picrotoxin, at 50 micromol l(-1), did not alter the ibotenate-induced reduction in contraction frequency, suggesting that this effect is not produced by inhibitory glutamate receptors. The glutamate-induced increase in tonus was antagonized by Joro spider toxin, JSTX-3. Thus, glutamate receptors associated with crayfish hindgut muscles are of the quisqualate type but are also sensitive to ibotenate. Elevating extracellular potassium concentration mimicked all of the effects of glutamate, suggesting that excessive depolarization may contribute to the suppression of contractions at high agonist concentrations.     

Inhibitory and excitatory mechanisms of neurotensin action in canine intestinal circular muscle in vitro.

The effect of neurotensin on canine ileal circular muscle devoid of myenteric plexus was investigated using single and double sucrose gap techniques. Similar results were obtained with microelectrode techniques. Neurotensin caused a temperature-sensitive and dose-dependent biphasic response, an initial hyperpolarization associated with inhibition of contractile activity, followed by an excitatory phase, usually consisting of spike discharge and tonic and phasic contractions, for which depolarization was not required. Neither response was affected by tetrodotoxin, phentolamine, propranolol, or atropine. The hyperpolarization was associated with decreased membrane resistance, blocked by 10(-7) M apamin, and converted to tonic depolarization by apamin (10(-6) M). Tachyphylaxis to neurotensin occurred when the stimulation interval was less than 20 min. After Ca2+ depletion, depolarization was observed instead of the hyperpolarization; this depolarization was not affected by nitrendipine and was gradually abolished with repetitive stimulation at 20-min intervals. When Ca2+ was present, nifedipine did not alter the hyperpolarizing phase of the response but inhibited spiking and blocked all contractions. The excitatory phase of the response was enhanced by Bay K-8644. Neuromedin N elicited a response identical with that of neurotensin. The responses of the two peptides were completely cross tachyphylactic. Inhibitory junction potentials were not affected by neurotensin tachyphylaxis. It is concluded that neurotensin and neuromedin N activate apamin-sensitive, calcium-dependent potassium channels in circular muscle, causing membrane hyperpolarization and inhibition of muscle contraction. Release of intracellular calcium is involved in the activation of these potassium channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stretch-activated ion channels contribute to membrane depolarization after eccentric contractions

We tested the hypothesis that eccentric contractionsactivate mechanosensitive or stretch-activated ion channels (SAC) in skeletal muscles, producing increased cation conductance.Resting membrane potentials and contractile function were measured in rat tibialis anterior muscles after single or multiple exposures to aseries of eccentric contractions. Each exposure produced a significantand prolonged (>24 h) membrane depolarization in exercised musclefibers. The magnitude and duration of the depolarization were relatedto the number of contractions. Membrane depolarization was dueprimarily to an increase in Na⁺ influx, because theestimated Na⁺-to-K⁺ permeability ratio wasincreased in exercised muscles and resting membrane potentials could bepartially repolarized by substituting an impermeant cation forextracellular Na⁺ concentration. Neither theNa⁺/H⁺ antiport inhibitor amiloride nor thefast Na⁺ channel blocker TTX had a significant effect onthe depolarization. In contrast, addition of either of two nonselectiveSAC inhibitors, streptomycin or Gd³⁺, produced significantmembrane repolarization. The results suggest that muscle fibersexperience prolonged depolarization after eccentric contractions due,principally, to the activation of Na⁺-selective SAC.

     

Prejunctional alpha 2 adrenoceptors inhibit contraction of tracheal smooth muscle by inhibiting cholinergic neurotransmission

Ring preparations obtained from the guinea pig trachea contracted on short trains of electrical field stimulation. These contractions were mediated by activation of cholinergic nerves since they were abolished by atropine or tetrodotoxin. In the presence of beta blocking drugs noradrenaline and adrenaline dose-dependently inhibited contractions induced by field stimulation. By contrast, contractions on exogenous acetylcholine were left completely unaffected. It is concluded that the adrenergic agonists inhibited cholinergic neurotransmission by a prejunctional action. In order to characterize the noradrenaline receptor the effects of alpha₁ and alpha₂ blockers were evaluated using the Schild plot. For comparison experiments were also conducted on the guinea pig aorta and electrically stimulated guinea pig ileum. The results indicate that in guinea pig trachea and ileum noradrenaline inhibits cholinergic neurotransmission by acting on prejunctional alpha₂ receptors whereas in guinea pig aorta it induces contraction by stimulating alpha₁ receptors.     

Activation of the fast calcium input in the denervated smooth muscle of the cat nictitating membrane

The excitation and contraction features of innervated and sympathetically denervated smooth muscle strips from cat's nictitating membrane have been studied by single sucrose gap arrangement. Increasing of smooth muscle cells sensitivity to drugs were accompanied by elevation of membrane response and the ability to generation of action potentials. Action potentials have been induced by agonists or high potassium concentration in external solution and spontaneously. In innervated muscle action potentials have been evoked as a result of depolarization by high potassium concentration of TEA blockade of potassium conductance. Induced and spontaneously generated action potentials were blocked by organic and inorganic antagonists of potential dependent Ca++ channels. In Ca-free solution action potentials were absent but might be supported by Ba++. Decrease of Na+ had no effect on smooth muscle excitability. It is supposed that activation of potential depended Ca++ channels in smooth muscle cells with pharmaco-mechanical coupling are under influence of sympathetic nerves.     

Neuromuscular block in locust skeletal muscle caused by a venom preparation made from the digger wasp Philanthus triangulum F. from Egypt.

A preparation from P. triangulum F., made by extracting abdomens and purified by Sephadex filtration, does not affect potassium ion-induced contractions of the retractor unguis muscle of S. gregaria, but the reduction of the glutamate contractions is at least as pronounced as the effect on the neurally-evoked twitch. Glutamate potentials are affected at a lower venom dose than are the neurally evoked excitatory postsynaptic potentials (EPSPs). The half-decay-time of the glutamate potentials starts to decrease just before the decrease in amplitude is initiated.In the retractor unguis muscle the resting plasma membrane is slightly depolarized at high venom concentrations, but this effect cannot explain the effects on neuromuscular transmission. It is concluded that the venom preparation of P. triangulum affects the glutamate or transmitter-induced transient permeability change, possibly by blocking the open ion-channels.     

Pharmacological reactions of the circular muscle of the guinea pig vas deferens.

Pharmacological responses of spiral strips prepared from the guinea pig vas deferens to various adrenergic and cholinergic agonists and autacoids were studied. On the circular muscle alpha adrenergic, muscarinic cholinergic and histaminergic receptors were identified. The responses evoked on the circular and longitudinal muscles were of the same type.     

Agonistic action of synthetic analogues of quisqualic acid at the insect neuromuscular junction

One hundred twenty analogues of quisqualic acid were synthesized and assayed on the neuromuscular junction of larva of the mealworm, Tenebrio molitor. Two new agonists for amino acid receptors, L-glutamic acid N-thiocarboxyanhydride (L-GANTA) and DL-hydantoinpropionic acid (DL-HPA), were discovered in this study. L-GANTA and DL-HPA produced muscle membrane depolarization, accompanied by a reduction of the muscle input resistance. The amplitude of excitatory postsynaptic potentials was decreased in the presence of L-GANTA and DL-HPA. The apparent dissociation constants obtained from dose-depolarization plots were 7 x 10^?4 M for L-GANTA and 9 x 10^?4 M for DL-HPA. Some structural constraints imposed on agonists at amino acid receptors on insect muscle were discussed.     

Actions of glutamate and ivermectin on the pharyngeal muscle of Ascaridia galli: a comparative study with Caenorhabditis elegans

The actions of glutamate and ivermectin were examined in the pharynx of Ascaridia galli and the results compared with those on the pharynx of Caenorhabditis elegans. In both preparations glutamate elicits a depolarization and inhibition of pharyngeal pumping, but the response of the pharynx of A. galli was much less than for C. elegans. This may be either because the pharyngeal membrane potential of the former is closely linked to the equilibrium potential for chloride ions (E(Cl)) while that of C. elegans is independent of E(Cl), or that there is a lower density of glutamate receptors on the pharyngeal muscle of A. galli compared with C. elegans. The maximum depolarization to glutamate of the pharyngeal muscle was 4.5+/-0.8 mV in A. galli while it was >25 mV in C. elegans. Picrotoxin was a weak antagonist of the glutamate response in both species. Flufenamic acid, pentobarbitone and flurazepam had no significant effect on either preparation at concentrations up to 100 microM. Three glutamate receptor agonists, ibotenate, kainate and quisqualate were all more potent than glutamate on the A. galli pharyngeal muscle. In contrast, only ibotenate was more potent than glutamate in C. elegans pharynx, the other two agonists being approximately 20 times less potent. The potency of ivermectin differed markedly between the two species, being approximately three orders of magnitude less potent on the pharynx of A. galli compared with C. elegans. This study demonstrates clear differences between the properties of the pharyngeal muscle of the two species and shows that care must be taken when extrapolating data from free-living to parasitic species of nematode.     

Distribution of chemoreceptors to quinine on the cell surface of Paramecium caudatum

The topological distribution of the chemoreceptors to quinine in the membrane of a ciliate Paramecium caudatum were examined by conventional electrophysiological techniques. A CNR-mutant specimen defective in voltage-gated Ca channels produced a transient depolarization followed by a transient hyperpolarization and a sustained depolarization when 1 mM quinine-containing solution was applied to its entirety. A Ni²⁺-paralyzed CNR-mutant specimen produced a simple membrane depolarization in response to a local application of 1 mM quinine-containing solution to its anterior end, whereas it produced a transient membrane hyperpolarization in response to an application to its posterior end. An anterior half fragment of a CNR specimen produced a membrane depolarization whereas a posterior half fragment of the specimen produced a transient hyperpolarization upon application of 1 mM quinine-containing solution. Both anterior depolarization and posterior hyperpolarization took place prior to the contraction of the cell body. It is concluded that Paramecium caudatum possesses two kinds of chemoreceptors or two kinds of coupling of the same receptor to different signal transduction pathways to quinine which are distributed in different locations on the cell surface. Activation of the anterior receptor produces a sustained depolarizing receptor potential while activation of the posterior receptor produces a transient hyperpolarizing receptor potential.Abbreviation CNR caudatum non reversal