Pharmacological sensitivity of the articular capsule of the primary spines of Eucidaris tribuloides

1. This paper describes the effects of several cholinergic agonists and antagonists, and of β-phenylethylamine (PEA) and some of its derivatives, on the articular capsule, or ligament, of the primary spines of Eucidaris tribuloides.2. Carbamylcholine (CCh), methacholine (MeACh), nicotine, and muscarine exert a stiffening effect similar to that of acetylcholine (ACh), although the time course of their actions varies widely.3. Atropine induced stiffening and blocked and responses to muscarine and MeACh. The responses to MeACh were blocked also by 4-diphenylacetoxy-N-methylpiperidine, suggesting the presence in the ligament of type M₃ muscarinic receptors, in addition to nicotinic ones. d-Tubocurarine induced stiffness of the ligament and failed to block the responses to ACh and nicotine.4. While ACh induced only a slight desensitization, CCh caused a long-lasting blockade of the stiffening effects of the cholinergic agonists. This shows that the receptors for ACh have a site or sites that recognize the ester moieties of these molecules.5. Eserine and neostigmine potentiate the responses to acetylcholine, indicating the presence of aeetyl-cholinesterase in the ligament.6. β-Phenylethy lamine, epinephrine, norepinephrine, and dopamine induce diphasic responses; usually a brief softening followed by a slow and irreversible stiffening of the ligament.7. In contrast to the above, tyramine and octopamine elicit a simple softening of ligaments which are stiff as a result of handling or by exposure to cholinergic agonists. However, tyramine and octopamine do not soften ligaments which become stiff as a result of exposure to adrenergic agonists.     

Effect of lignocaine on intestinal smooth muscle of rat ileum

The effect of lignocaine on tone and contractility of intestinal smooth muscle, and on contractures produced by ACh or TEA, was studied in isolated ileum of the rat. Lignocaine (0.1-100 microM) produced concentration-dependent contractures in the rat ileum. In low concentrations, lignocaine increased the amplitude of spontaneous contractions and contractions produced by transmural stimulation. High concentrations of lignocaine abolished all contractile responses and produced a marked contracture in rat ileum. Lignocaine (10 microM) also reduced the contractures produced by ACh (0.01-10 microM). In contrast, the contractures produced by TEA (0.1-10 mM) were markedly increased by lignocaine. Furthermore, the contracture produced by lignocaine was reduced by lowering the external calcium from 2.5 mM to 1.5 mM. It was concluded that lignocaine in moderate and high concentrations produces a contracture in rat intestinal smooth muscle. Whereas lignocaine reduces the ACh-induced contracture, it increases that produced by TEA in the same preparation. The results further suggest that lignocaine modifies cholinergic responses and affects excitation-contraction coupling in rat intestinal smooth muscle.     

Extracellular calcium dependence of contracture and modulation by serotonin in buccal muscle E1 of Aplysia

Serotonin [5-hydroxytryptamine (5-HT)] enhances acetyl choline (ACh)-elicited contractures of Aplysia buccal muscles E1 and I5. The possible role of external calcium in regulating the magnitude of ACh contracture in the presence and absence of 5-HT was investigated. Superfusion of E1 with zero calcium medium caused ACh contractures to fail within one to two minutes. Recovery of ACh contracture upon restoring normal medium occurred within two to four minutes. In the absence of 5-HT, ACh contracture decreased proportionally to external [Ca⁺⁺] in the concentration range of 0–10 mM; however, the amount of enhancement of of ACh contracture following 5-HT treatment did not decrease with external [Ca⁺⁺] as long as [Ca⁺⁺] was above a threshold concentration that varied from preparation to preparation. For most preparations, the enhancement of ACh contracture by 5-HT was dependent on the presence of external calcium during 5-HT treatment. Calcium influx into muscles E1 and I5 increased approximately two and a half fold in the presence of 10^?6 M 5-HT. A model in which 5-HT brings about calcium “loading” of an ACh releasable intracellular storage site is discussed.     

Cholinergic control of the mechanical properties of the catch connective tissue in the holothurian body wall

Effects of acetylcholine (ACh), ACh-agonists and antagonists were studied on the viscosity of the dermis of the sea cucumber Holothuria leucospilota. ACh and nicotinic agonists caused an early increase in viscosity and late decrease. Muscarinic agonists produced a viscosity decrease. The viscosity increase elicited by nicotine was inhibited by tubocurarine. The viscosity decrease caused by methacholine was suppressed by atropine. The mechanical properties of this connective tissue are very likely controlled by both nicotinic and muscarinic cholinoreceptors.     

Acetylcholine sensitivity of the spine-test articular capsule of the sea urchin Evcidaris tribuloides

1. The changes in the consistence of the spine-test articular capsule, or ligament, of the primary spines of Eucidaris tribuloides induced by acetylcholine (ACh) have been studied. Two complementary techniques were used: (a) “forced-vibration”, which detects variations in the stiffness of the ligament along a single diametral plane; and (b) “forced-rotation” which records the spatial distribution of those changes.2. ACh (1 μM to 1 mM) caused a rapid increase in the resistive force opposed by the ligament to passive stretching. Similar effects were elicited by several monoquaternary, N-substituted derivatives of trimethylammonium.3. The opposite effect, i.e. softening, was induced by decamethonium, dimethylphenylpiperazine, and 2-ketoamyltrimethylammonium.4. The involvement in these effects of ACh-binding groups with pharmacological properties similar to those of the “anionic sites” of nicotinic ACh receptors is suggested.     

Cholinergic transmission at mechanosensory afferents in the crayfish terminal abdominal ganglion

Electrical stimulation of mechanosensory afferents innervating hairs on the surface of the exopodite in crayfish Procambarus clarkii (Girard) elicited reciprocal activation of the antagonistic set of uropod motor neurones. The closer motor neurones were excited while the opener motor neurones were inhibited. This reciprocal pattern of activity in the uropod motor neurones was also produced by bath application of acetylcholine (ACh) and the cholinergic agonist, carbamylcholine (carbachol). The closing pattern of activity in the uropod motor neurones produced by sensory stimulation was completely eliminated by bath application of the ACh blocker, d-tubocurarine, though the spontaneous activity of the motor neurones was not affected significantly. Bath application of the acetylcholinesterase inhibitor, neostigmine, increased the amplitude and extended the time course of excitatory postsynaptic potentials (EPSPs) of ascending interneurones elicited by sensory stimulation. These results strongly suggest that synaptic transmission from mechanosensory afferents innervating hairs on the surface of the tailfan is cholinergic.Bath application of the cholinergic antagonists, dtubocurarine (vertebrate nicotinic antagonist) and atropine (muscarinic antagonist) reversibly reduced the amplitude of EPSPs in many identified ascending and spiking local interneurones during sensory stimulation. Bath application of the cholinergic agonists, nicotine (nicotinic agonist) and oxotremorine (muscarinic agonist) also reduced EPSP amplitude. Nicotine caused a rapid depolarization of membrane potential with, in some cases, spikes in the interneurones. In the presence of nicotine, interneurones showed almost no response to the sensory stimulation, probably owing to desensitization of postsynaptic receptors. On the other hand, no remarkable changes in membrane potential of interneurones were observed after oxotremorine application. These results suggest that ACh released from the mechanosensory afferents depolarizes interneurones by acting on receptors similar to vertebrate nicotinic receptors.Abbreviations ACh cetylcholine - mns motor neurones - asc int ascending interneurone     

Dual muscarinic and nicotinic action on a motor program in Drosophila

The effect of cholinergic agonists and antagonists on the central pattern generator of the pharyngeal muscles has been studied in third instar larvae of Drosophila. The pharyngeal muscles are a group of rhythmically active fibers involved in feeding. Bath application of the cholinergic agonists carbachol, musarine, pilocarpine, and acetylcholine (ACh) to a semiintact preparation including the pharyngeal muscles and the central nervous system (CNS), initiated long-lasting endogenous-like bursting activity in the muscles. The muscarinic antagonists, atropine and scopolamine, blocked these responses as well as endogenous activity. Perfusion with nicotine elicited a short, tonic response that was marginally blocked by mecamylamine but not by curare, α-bungarotoxin, hexamethonium, or the muscarinic antagonists. This is the first time that a response to cholinergic drugs has been examined in Drosophila. The pharyngeal muscle preparation may prove to be a valuable system for studying mutations of cholinergic metabolism, receptors, and second messengers.     

Carbachol contracture of stomach smooth muscle of the newt in calcium-free solution

A small muscle preparation of stomach circular muscle of the newt responded to carbachol (CCh) with a phasic contracture. At 20 degrees C, in Ca-free Ringer solution (+1 mM EGTA), the amplitude of CCh contracture was very rapidly inhibited to less than 10% of that in normal Ringer solution (1.8 mM Ca). The amplitude of this CCh contracture was markedly enhanced with increasing [K]0. CCh contracture in Ca-free Ringer solution was also enhanced after K contracture was induced once in the presence of 1.8 mM Ca, followed by soaking in normal Ringer solution. The amplitude of this enhanced CCh contracture persisted up to about 5 min, following rapid decrease to about 70%, and then gradually decreased to a steady level in Ca-free Ringer solution. This decrease in amplitude was prevented by increasing [K]0 during soaking in Ca-free solution; even when the temperature was elevated from 20 to 35 degrees C during the periods of soaking in Ca-free solution, CCh contracture was inhibited only by about 20% in Ca-free high K solution, whereas in Ca-free or Ca-free low Na (Tris) Ringer solution it was inhibited by more than 50%.     

Dual muscarinic and nicotinic action on a motor program in Drosophila.

The effect of cholinergic agonists and antagonists on the central pattern generator of the pharyngeal muscles has been studied in third instar larvae of Drosophila. The pharyngeal muscles are a group of rhythmically active fibers involved in feeding. Bath application of the cholinergic agonists carbachol, muscarine, pilocarpine, and acetylcholine (ACh) to a semiintact preparation including the pharyngeal muscles and the central nervous system (CNS), initiated long-lasting endogenous-like bursting activity in the muscles. The muscarinic antagonists, atropine and scopolamine, blocked these responses as well as endogenous activity. Perfusion with nicotine elicited a short, tonic response that was marginally blocked by mecamylamine but not by curare, alpha-bungarotoxin, hexamethonium, or the muscarinic antagonists. This is the first time that a response to cholinergic drugs has been examined in Drosophila. The pharyngeal muscle preparation may prove to be a valuable system for studying mutations of cholinergic metabolism, receptors, and second messengers.     

Effects of Cannabinoids on Tension Induced by Acetylcholine and Choline in Slow Skeletal Muscle Fibers of the Frog

We investigated the effects of cannabinoids on acetylcholine (ACh) or choline contractures in slow skeletal muscle fibers from Rana pipiens. Bundles of cruralis muscle fibers were incubated with the cannabinoid receptor 1 (CB₁) agonist, arachidonylcyclopropylamide (ACPA), which diminished the maximum isometric tension by 10 % and the total tension by 5 % of the ACh contracture, and 40 and 22 % of the choline contracture, respectively. Preincubation with the CB₁ antagonist, AM281, or with pertussis toxin (PTX) completely blocked the effect of ACPA on the ACh contracture. On the other hand, the decrease in choline contracture by ACPA was only partially blocked by AM281 (~16 % decrease), PTX (20 %), or by dantrolene (~46 %). Our results show that ACPA modulates ACh and choline contractures, and suggest that this effect involves the participation of CB₁, the ACh receptor, and ?RyR in ACh contractures. For choline contractures, ACPA may also be acting through cannabinoid receptor-independent mechanisms.     

The nature of the acetylcholine and 5-hydroxytryptamine receptors in buccal smooth muscle of the pest slug Deroceras reticulatum

The characteristics of the acetylcholine (ACh) and 5-hydroxytryptamine (5-HT) receptors of Deroceras buccal muscle were examined using specific pharmacological probes and sucrose gap electrophysiological analysis. ACh induced concentration-dependent smooth tonic contractures coupled with considerable depolarisation from the normal resting membrane potential of -30.6 mV. The use of choline ester analogues such as carbachol, propionylcholine and butyrylcholine, specific cholinergic agonists such as nicotine, muscarine, bethanecol and pilocarpine and antagonists such as d-tubocurarine, succinylcholine, hexamethomium, atropine, gallamine, pirenzepine and scopolamine indicated that the ACh receptor showed both nicotinic and muscarinic characteristics; the muscarinic activity resembled that of a mammalian M(2)-like receptor. Alternatively, it can not be ruled out that both mammalian types of receptor may be present in this preparation since both nicotine and muscarine induced noticeable tension. 5-HT application induced characteristic dose-dependent phasic contractions accompanied by small but quite consistent depolarisations. Serotonergic agonist and antagonist experiments using 1-(3-chlorophenyl) piperazine, 1-(m-chlorophenyl) biguanide, methiothepin, methysergide and metoclopramide strongly suggested that the 5-HT receptor showed closest pharmacological affinity with the 5-HT(1) receptor class of mammals but with some 5-HT(2) activity. In view of the phylogenetic gap between molluscs and mammals it is not surprising that the ACh and 5-HT receptors of Deroceras can not be properly classified by conventional mammalian terminology.     

Identification of acetylcholine and impact of cholinomimetic drugs on cell differentiation and growth in the unicellular green alga Micrasterias denticulata

Acetylcholine (ACh), one of the best-studied neurotransmitters, has been reported in animals as well as in multicellular plants. In higher plants, ACh affects phytochrome-dependent growth and differentiation. In the present study on the green alga Micrasterias denticulata which has been used as a cell biological model system since several decades, we identified ACh for the first time in a unicellular alga, demonstrated light as a regulatory factor of ACh production and showed that cholinergic agonists and antagonists suppress growth and differentiation. ACh was detected in Micrasterias cells grown under light–dark conditions of 14/10 h, but not in dark-grown algae, by high-performance liquid chromatography coupled with mass spectrometry. To quantify cholinergic effects on cell differentiation, we exposed young developmental stages of Micrasterias to cholinergic antagonists (d-tubocurarine, hexamethonium) as well as agonists (carbachol, nicotine). We found that the cholinergic antagonists and, surprisingly, the agonist nicotine significantly suppressed cell growth and differentiation in a dose-dependent manner. Moreover, we demonstrated that secondary wall formation is specifically inhibited in the presence of nicotine.     

Cholinergic transmission from mechanosensory afferents to an identified nonspiking interneuron in the crayfish Procambarus clarkii girard

Pharmacological properties of excitatory synaptic transmission from mechanosensory afferents to an identifiable nonspiking interneuron of crayfish were studied by drug perfusion experiments using acetylcholine (ACh) agonists and antagonists. Application of carbachol, a general agonist of ACh, caused sustained depolarization of the interneuron and a decrease in the peak amplitude of its excitatory synaptic response to sensory stimulation on the soma side. Similar depolarization was observed during application of carbachol under the low-Ca²⁺, high-Mg²⁺ condition. The peak amplitude was also reduced by application of nicotine and tetramethylammonium, both of which also caused sustained depolarization of the inter-neuron. By contrast, perfusion of muscarinic agonists, muscarine, oxotremorine and pilocarpine, reduced the peak amplitude without affecting the membrane potential of the interneuron. Perfusion of nicotonic antagonists of ACh, d-tubocurarine and hexamethonium, caused reduction of the peak amplitude without any change in the membrane potential. A muscarinic antagonist atropine was also effective in blocking the synaptic transmission but at higher concentration than d-tubocurarine. The results suggest that the ACh receptors on the nonspiking interneuron belong to a previously characterized class of crustacean cholinergic receptors resembling the nicotinic subtype of vertebrates.     

Expression and function of neuronal nicotinic ACh receptors in rat microvascular endothelial cells

The expression and function of nicotinic ACh receptors (nAChRs) in rat coronary microvascular endothelial cells (CMECs) were examined using RT-PCR and whole cell patch-clamp recording methods. RT-PCR revealed expression of mRNA encoding for the subunits alpha(2), alpha(3), alpha(4), alpha(5), alpha(7), beta(2), and beta(4) but not beta(3). Focal application of ACh evoked an inward current in isolated CMECs voltage clamped at negative membrane potentials. The current-voltage relationship of the ACh-induced current exhibited marked inward rectification and a reversal potential (E(rev)) close to 0 mV. The cholinergic agonists nicotine, epibatidine, and cytisine activated membrane currents similar to those evoked by ACh. The nicotine-induced current was abolished by the neuronal nAChR antagonist mecamylamine. The direction and magnitude of the shift in E(rev) of nicotine-induced current as a function of extracellular Na(+) concentration indicate that the nAChR channel is cation selective and follows that predicted by the Goldman-Hodgkin-Katz equation assuming K(+)/Na(+) permeability ratio of 1.11. In fura-2-loaded CMECs, application of ACh, but not of nicotine, elicited a transient increase in intracellular free Ca(2+) concentration. Taken together, these results demonstrate that neuronal nAChR activation by cholinergic agonists evokes an inward current in CMECs carried primarily by Na(+), which may contribute to the plasma nicotine-induced changes in microvascular permeability and reactivity induced by elevations in plasma nicotine.     

Effects of cholinergic agonists on diacylglycerol and intracellular calcium levels in pancreatic β-cells

We have studied the effects of cholinegic agonists on the rates of insulin release and the concentrations of diacylglycerol (DAG) and intracellular free Ca²⁺ ([Ca²⁺]_i) in the β-cell line MIN6. Insulin secretion was stimulated by glucose, by glibenclamide and by bombesin. In the presence of glucose, both acetylcholine (ACh) and carbachol (CCh) produced a sustained increase in the rate of insulin release which was blocked by EGTA or verapamil. The DAG content of MIN6 β-cells was not affected by glucose. Both CCh and ACh evoked an increase in DAG which was maximal after 5 min and returned to basal after 30 min; EGTA abolished the cholinergic-induced increased in DAG. ACh caused a transient rise in [Ca²⁺]_i which was abolished by omission of Ca²⁺ or by addition of devapamil. Thus, cholinergic stimulation of β-cell insulin release is associated with changes in both [Ca²⁺]_i and DAG. The latter change persists longer than the former and activation of protein kinase C and sensitization of the secretory process to Ca²⁺ may underlie the prolonged effects of cholinergic agonists on insulin release. However, a secretory response to CCh was still evident after both [Ca²⁺]_i and DAG had returned to control values suggesting that additional mechanisms may be involved.     

Cholinergic receptors and catecholamine secretion from adrenal chromaffin cells of the toad

1. The effects of cholinergic drugs on catecholamine (CA) secretion from adrenal chromaffin tissue of the toad were studied.2. CA secretion was induced by ACh or nicotine, but not by muscarine.3. Hexamethonium inhibited the CA release evoked by ACh or nicotine, while d-tubocurarine only affected the nicotinic response. Atropine did not prevent the secretory response.4. Muscarine abolished the secretion induced by the agonists, this effect being prevented by atropine or gallamine, but not by pirenzepine.5. In conclusion, CA secretion in the toad is stimulated by activation of nicotinic receptors. Inhibitory muscarinic receptors are present, most likely of type M₂, which may play a regulatory function.     

Nicotinic acetylcholine receptors on a cholinergic nerve terminal in the cockroach,Periplaneta americana

Summary Intracellular microelectrode recording and ionophoretic application of carbamylcholine (CCh) were used to compare the cholinergic sensitivity of postsynaptic dendrites of an identified neurone with that of an identified presynaptic cholinergic axon.The axon of the lateral filiform hair sensory neurone (LFHSN) in the first-instar cockroachPeriplaneta americana was found to be as sensitive to CCh as the dendritic regions of giant interneurone 3 (GI 3). The CCh response of both neurones was unaffected by replacing Ca²⁺ with Mg²⁺, confirming that the ACh receptors are present on the neurones under test. The CCh response of both neurones was mimicked by ionophoretic application of nicotine. The responses were blocked by 10^–5 M mecamylamine and 10^–6 M d-tubocurarine and were not affected by muscarinic antagonists, suggesting that the ACh receptors present on GI 3 and LFHSN are predominantly nicotinic.The muscarinic agonist oxotremorine and the antagonists atropine and quinuclidinyl benzilate had no modulatory effect on LFHSN-GI 3 synaptic transmission.The latency of the LFHSN response to CCh was consistent with the hypothesis that ACh receptors are situated on the main axon/terminal within the neuropil of the ganglion. It has previously been shown that this region of the axon does not form output synapses (Blagburn et al. 1985a). This indirect evidence indicates that presynaptic or extrasynaptic ACh receptors are present in the membrane of a cholinergic axon.LFHSN was depolarized by synaptically-released ACh after normal or evoked spike bursts, suggesting that the nicotinic ACh receptors act as autoreceptors. However, it was not possible to obtain direct evidence to support the hypothesis that these receptors modulate ACh release.Abbreviations CCh carbamylcholine - GI giant interneurone - FHSN filiform hair sensory neurone - LFHSN lateral filiform hair sensory neurone - R _in input resistance - V depolarization - V _m resting potential     

Excitatory effects of cholinergic,adrenergic and glutaminergic agonists on a buccal muscle of Aplysia

The anterior extrinsic protractors in the buccal mass of Aplysia are symmetrical sheets of branching bundles of muscle fibers which form an electrical syncytium. The addition of potassium or cholinergic, adrenergic, and glutaminergic agonists to the sea water bathing medium produces contracture of the muscle. Strychnine and cholinergic or adrenergic antagonists all block contractures produced by cholinergic and adrenergic agonists but not those produced by potassium or glutamate. Iontophoretic application through microelectrodes of acetylcholine or dopamine anywhere on the muscle surface produced a graded depolarization of the membrane. By contrast, glutamate produces depolarization only at discrete membrane sites. Endogeneous contractions often appeared spontaneously or could be induced by drug exposure. ATP inhibits endogeneous contractions.     

Do independent processes control the activation and inactivation of potassium contracture tension in rat skeletal muscle?

Potassium (K⁺) contracture tension, measured in small bundles of rat soleus muscle fibers during maintained depolarization, increases to a peak value and then decays either to the baseline or to a pedestal level. We have tested the hypothesis that the rise and fall of tension are determined by independent activation and inactivation processes. If the “Independence” hypothesis is correct, tension during the decay of K⁺ contractures should equal tension predicted from the product of the activation and inactivation parameters determined from the same K⁺ contractures. Both the measured and predicted tensions decayed to a pedestal level that was increased in amplitude in the presence of perchlorate ions. However, the measured tensions in normal solutions and in the presence of perchlorate were three to five times smaller than the predicted tensions. This result indicates that the activation and inactivation of processes controlling the rise and decay of K⁺ contracture tension are not independent.     

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.