Acetylcholine Sensitivity Research Articles

Cholinergic excitation and inhibition in the visual thalamus of the cat — influences of cortical inactivation and barbiturate anesthesia

Neuronal responses to microiontophoretically applied pulses of acetylcholine (ACh) were recorded in the perigeniculate nucleus (PGN) and dorsal lateral geniculate nucleus (dLGN) of the cat. In the PGN, ACh inhibited 95% of the cells under N 2O/O 2-halothane anesthesia. Systemic application of sodium pentobarbitone did not abolish ACh inhibitio. In the dLGN with N 2O-halothane, ACh increased firing rates of neurons before (88%) and after (78%) interruption of their direct retinal inputs by photocoagulation of the receptive field area on the retina. Functional inactivation of the corticogeniculate loop by cooling of the ipsilateral visual cortex did not selectively influence ACh sensitivity in the retinally deafferented dLGN. It is concluded that ACh exerts a direct, excitatory action on geniculate neurons. Infusion of sodium pentobarbitone resulted in reduction of ACh firing rates to about 30% of the control values in 35% of the normally innervated cells (65% of that sample not being excited by ACh), whereas excitatory ACh responses after retinal deafferentation were completely blocked by the barbiturate. It is concluded that pentobarbitone influences direct ACh excitation, but not cholinergic disinhibition in the dLGN or ACh inhibition in the PGN. Possible differential effects of pentobarbitone on nicotine and muscarinic types of ACh responses are discussed.

Muscarinic stimulation of submucosal glands in swine trachea.

The properties of muscarinic acetylcholine receptors (mAChR) on tracheal explants and isolated submucosal gland cells were determined using [3H]quinuclidinyl benzilate ([3H]QNB) and N-[3H]methylscopolamine ([3H]NMS) as ligands. Analysis of competitive displacement of ([3H]NMS binding by pirenzepine demonstrated the presence of M1- (27 +/- 2%) and M2G- (73 +/- 2%) receptors on isolated tracheal submucosal gland cells (TSGC's) in control. Daily administration of diisopropylfluorophosphate (DFP) inhibited cholinesterase activity by greater than 95%. After 7 days of DFP treatment, [3H]QNB binding to intact TSGC's decreased from 14.2 +/- 0.6 to 6.3 +/- 0.8 fmol/10(6) cells; similarly, [3H]NMS binding fell from 8.1 +/- 1.9 to 2.0 +/- 0.8 fmol/10(6) cells. The loss of mAChR's was predominantly of the M2G subtype with the relative proportion dropping to 33%. In addition, 90% of the receptors assumed the high-affinity state for carbachol displacement of [3H]NMS. Mucus secretion was quantitated by measuring the release of 3H-labeled mucus macromolecules from explants of tracheal submucosal glands and isolated cells. Acetylcholine (ACh), 2 X 10(-5) M, stimulated mucus secretion by 2.5 and 2.3 times the basal rate, respectively. Elimination of acetylcholinesterase (AChe) by DFP increased the ACh sensitivity by 18- and 5-fold. Tracheal explants or TSGC's obtained 2 h after an in vivo DFP treatment showed a 6- and 3-fold ACh stimulation. This ACh sensitivity decreased during the continued daily dosing with DFP such that only a 1.3- and 1.1-fold ACh stimulation was apparent after 7 days of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of ?-endorphin on the development of denervation changes in rat muscle fiber membrane

Recordings were made of post-denervation changes in resting potential and input resistance in muscle fiber membrane, as well as anode break, tetrodotoxin resistant action potentials, and asynaptic sensitivity to acetylcholine during experiments on cultured diaphragm muscle fiber isolated from rats. Addition of β-endorphin to the culture medium prevented increase in the input resistance of muscle fibers and reduced development of asynaptic transmitter sensitivity in the membrane, but failed to change the ability of the denervated muscle membrane to generate anode break and tetrodotoxin-resistant action potentials. The effects of β-endorphin were not abolished by naloxone, which itself had endorphin-like powers as measured by the indices used in this research. It is therefore suggested that β-endorphin or like substances could be claimed as the neurotrophic factors responsible for controlling passive electrical properties of the muscle fiber membrane and contribute to regulating its acetylcholine sensitivity.

The effect of anticholinesterase drugs on the ionophoretically-evoked end-plate currents.

In order to study the sensitivity of acetylcholine (ACh) receptors exposed to anticholinesterase drugs, the ionophoretically evoked end-plate currents (e.p.c.i) in frog sartorius muscle were studied. During treatment with prostigmine (PST, 3 microM) or methanesulfonyl fluoride (MSF, 1-2 mM), the amplitude of the e.p.c.i evoked by ACh was first increased, reaching its maximum (200-400%) after 20-30 min and then decreased. If these experiments were repeated by applying carbachol (CCh) instead of ACh, an increase, although less pronounced (30-100%), was also observed. The results obtained by applying CCh cannot be ascribed to the inhibition of ACh-esterase, as CCh is not hydrolysed by this enzyme. Therefore, a direct action of both drugs on the ACh-receptor channel complex is suggested. From the comparison of the effects of PST and MSF on the responses to CCh and ACh applications, it is calculated that 20-25% of the increased response to ACh during PST treatment can be ascribed to the direct effect on the ACh-receptor channel complex. During MSF treatment, this amount results in 13-15%. The changes of the time course of the e.p.c.i (evoked by CCh or ACh), which is prolonged in the presence of PST and shortened during the maximum effect of MSF, suggest that the direct action of both studied drugs is different.

Ruthenium red reduces acetylcholine sensitivity and increases desensitization at the frog neuromuscular junction

The actions of Ruthenium Red on the synaptic membrane were studied at the neuromuscular junction of the frog Rana pipiens. Ruthenium Red blocks mitochondrial calcium transport and thus is expected to elevate the intracellular calcium level [ Alnaes and Rahamimoff (1975) J. Physiol., Lond. 248, 285–306]. The postjunctional membrane sensitivity was reduced by Ruthenium Red as determined by (a) the amplitude of the miniature endplate potentials, (b) iontophoretic application of carbachol, (c) microperfusion of carbachol. Desensitization was assayed by measuring the decline in the amplitude of the postsynaptic depolarization produced during repetitive iontophoretic application of carbachol. Following Ruthenium Red treatment desensitization was increased. This action of Ruthenium Red was enhanced by raising the extracellular calcium concentration from 1.8 to 10 mM.

Effects of pyrethroids on nicotinic acetylcholine receptor binding and function

The mechanism of interaction of two pyrethroids with the nicotinic acetylcholine (ACh) receptor was investigated by means of their effects on binding of radioligands to Torpedo electric organ receptor and its tracer ion flux, as well as by means of their effects on ACh sensitivity of denervated rat soleus muscle. Allethrin and fluvalinate had no effect on the equilibrium binding of 125I-labeled α-bungarotoxin (α-BGT) to the ACh receptor, but both decreased its initial rate of binding in presence of carbamylcholine (carb). In absence of carb, only allethrin decreased the rate of 125I-α-BGT binding slightly. Binding of [ 3H]perhydrohistrionicotoxin (H 12-HTX) to the channel sites of the ACh receptor was inhibited more by allethrin than by fluvalinate, but unlike H 12-HTX, neither pyrethroid had much effect on receptor-activated 22Na + influx into Torpedo microsacs, nor did fluvalinate reduce ACh sensitivity in denervated rat soleus muscle. Thus, it is suggested that the pyrethroids bind to a site that is different from the agonist or H 12-HTX binding sites. Both fluvalinate and allethrin were potent in inhibiting receptor-activated 45Ca 2+ uptake, and their effect increased with time. The RS fluvalinate isomer was more potent than the RR in inhibiting 45CA 2+ uptake, in agreement with their relative toxicities to insects. Fluvalinate and allethrin (1 μ m) increased the affinity of ACh receptors for carb at low concentrations and decreased it at high concentrations, suggesting that binding of pyrethroids affects agonist-induced conformational changes. The ACh sensitivity of denervated soleus muscle fibers was greatly depressed when the buffer was Na + free and contained 95 m m Ca 2+, but fluvalinate significantly increased the ACh sensitivity. Thus, it is suggested that the pyrethroids bind to the nicotinic ACh receptor and interfere with receptor desensitization.

Electrophysiologic denervation changes of human muscle fibers in motoneuron diseases.

Muscle denervation and reinnervation by sprouting of the surviving motoneurons characterizes motoneuron diseases (MND). In mammalian muscles, experimental denervation induces the appearance of extrajunctional acetylcholine (ACh) receptors and tetrodotoxin (TTX) resistant action potentials (AP). These denervation changes have been investigated in muscle biopsies from 10 MND patients and in 2 traumatically denervated normal human muscles. Extrajunctional ACh sensitivity was present in 113 of the 140 (73%) fibers from MND muscles studied. In 50 of 84 (70%) ACh-sensitive fibers, no TTX-resistant AP were present. The remaining fibers (30%) showed small regenerative responses. In contrast, all the traumatically denervated muscle fibers showed extrajunctional ACh sensitivity and TTX-resistant AP. Histochemical analysis of the biopsies showed no direct correlation between the frequency of ACh-sensitive fibers and that of the atrophic or normal-appearing fibers. The absence of TTX-resistant AP in ACh-sensitive fibers and its lack of correlation with the histochemical criteria of denervation suggest the presence of a state of innervation in MMD, where the motoneuron is not able to maintain its fully trophic influence on the muscle fiber membrane.

The relationship between acetylcholine- and histamine-induced constriction of large airways in normal subjects and subjects with asthma: A possible role for postreceptor mechanisms

Whether in man histamine (H) and acetylcholine (ACH) airway responses are interrelated is controversial. With the use of quantitative nebulization and specific airway conductance ( G aw V L , we recorded noninvasive pharmacologic tests of H- and ACH-induced bronchoconstriction in 11 normal subjects (N) and nine subjects with asthma (AST) with and without pretreatment of 0.75 mg (A 0.75) and 1.50 mg (A 1.50) of inhaled atropine. Provocation dose 40 (PD 40) for H or ACH were different in N (0.8 × 10 −2 mol/L and 2.9 × 10 −2 mol/L, respectively) and AST (5.0 × 10 −3 mol/L and 7.8 × 10 −3 mol/L) but linearly related. The ratio PD 40-ACH PD 40-H was comparable in N and AST and similar to the ratio ACH sensitivity/H sensitivity found in vitro for large airways. The slopes of log dose-response curves (SLDRC) to H and SLDRC to ACH were of similar magnitude both in N and AST and linearly related; however, for either agonist, bronchial sensitivity (PD 40) and reactivity (SLDRC) failed to correlate. After A 0.75, the dose ratios (DR) of H and ACH were large, with substantial intersubject variability, and similar in N (4.32 and 9.54, respectively) and AST (5.97 and 5.64). Although numerically comparable, DR of H and DR of ACH were unrelated. After A 1.50, DR of H remained unchanged (4.53 in N and 5.44 in AST), but DR of ACH further increased (17.37 in N and 8.13 in AST). Pretreatment with A 0.75 respected the linear relationship of PD 40-H versus PD 40-ACH but blurred that of reactivity to these agonists. After A 0.75 or A 1.50, solΔG aw V L was similar in N and AST. Except for PD 40, none of the tests recorded could distinguish AST from N. In conclusion, H-induced bronchoconstriction is substantially but not exclusively mediated via ACH pathway. We hypothesize that H and ACH responses anastomose within the cholinergic pathway at a sensitivity and a reactivity level; the former level is ostensibly downstream from and unrelated to the latter. In AST, the reactivity to H and ACH, the bronchodilating, anti-histaminic (DR of H) and antimuscarinic (DR of ACH) effects of atropine are normal and so is ostensibly the anastomosis between H and ACH reactivity levels. The level of H and ACH sensitivity distinguishes AST from N. ACH-sensitivity site may be located at the H->ACH anastomosis.

A muscle cell line from dystrophic mice expressing an altered phenotype in vitro

The isolation and characterization of a myogenic cell line from C57BL/6J/dydy mice is described. This line (DyA4) maintains the morphological, biochemical and elec-trophysiological characteristics of the primary cultured cells, at least for 20 passages. The cells actively divide as long as they are subcultured in media supplemented with horse serum and embryo extract. If the cells are not subcultured for a few days, they fuse into multinucleated contracting myotubes, which readily synthesize specific muscle products such as acetylcholinesterase and acetylcholine receptor. This dystrophic cell line expresses in vitro the same altered phenotype that is characteristic of dystrophic muscle cells in primary cultures, namely reduced acetylcholine sensitivity and reduced acetylcholine receptor expression. Because they can be grown in large amounts, and represent a pure muscle cell population which express an altered phenotype in an in vitro aneural avascular environment, DyA4 cells provide a very useful model system for investigating the pathogenesis of murinc muscular dystrophy.

Factors affecting the expression of acetylcholine receptors on rat sensory neurones in culture.

Sensory neurones from nodose ganglia of new-born rats were grown in dissociated tissue culture either with or without satellite cells. When cultured without satellite cells, most neurones developed sensitivity to acetylcholine (ACh); time-course experiments indicated that the neurones acquire their sensitivity during the second to third week in culture. Most neurones co-cultured with satellite cells did not develop ACh sensitivity. Delayed removal of satellite cells 8-12 days after plating resulted in few neurones acquiring ACh sensitivity. Delayed addition of satellite cells to neuronal cultures that were initially grown without satellite cells had no effect on the number of ACh-sensitive neurones. The potential to develop ACh sensitivity in culture without satellite cells decreases with the age of the neurones at the time of culturing; few neurones from 2-week-old animals developed sensitivity to ACh when cultured without satellite cells. These results indicate that there is some influence from satellite cells that prevents nodose neurones from developing ACh sensitivity in culture and suggests that this influence may also operate in vivo.

Pancreatic adaptation in VMH obesity: in vivo compensatory response to altered neural input.

The objective of these studies was to determine the impact of enhanced insulin response to glucose (glucose responsiveness) and altered neurotransmitter sensitivity of the pancreatic islet previously observed after ventromedial hypothalamic (VMH) lesion on the glucose regulatory system in the whole animal. Female Wistar (220 g) rats received bilateral electrolytic VMH lesions and were implanted with cardiac and femoral cannulas under anesthesia. After recovery, experimental studies were conducted in 2-h-fasted lightly heparinized conscious rats. Plasma glucose declined at 1 and 2 wk after lesion and returned gradually to basal levels by 4 wk, whereas plasma insulin was elevated at 1 wk and remained elevated at 4.5 wk after lesion. Glucose responsiveness was increased threefold 1 wk after lesion. Acetylcholine sensitivity, measured by insulin response to an acetylcholine analogue, was decreased at 1, 2, and 4 wk after lesion, whereas norepinephrine sensitivity, measured by norepinephrine inhibition of the insulin response to intravenous glucose, was significantly increased 1 wk after VMH lesion. These studies suggest that the observed alterations at the level of the pancreatic islet led to a shift in the operating point of the glucose regulatory system of the whole animal and that changes in neurotransmitter sensitivity reflect a pancreatic adaptation subsequent to the establishment of enhanced glucose responsiveness. This shift in operating point may be secondary to altered neural input and may be essential to pancreatic and, thus, metabolic adaptation to the VMH lesion-induced obese state.

Enkephalin and substance p modulate synaptic properties of chick ciliary ganglion neurons in cell culture

Since enkephalin- and substance P-like immunoreactive materials have been identified in preganglionic terminals of the avian ciliary ganglion, we tested the effects of enkephalin and substance P directly on chick ciliary ganglion neurons in dissociated cell culture. Under these conditions the neurons form cholinergic synapses with each other that are spontaneously active. Both peptides modulate properties of membrane components associated with synaptic transmission between the neurons. Enkephalin causes a 60% reduction in the mean amplitude of the excitatory synaptic potentials, and the effect appears to be presynaptic in origin: enkephalin does not alter acetylcholine sensitivity on the neurons, but does inhibit Ca 2+ influx as reflected by a 38% shortening of the Ca 2+ component of the action potential. Both the reduction in synaptic potential amplitude and the shortening of the Ca 2+ action potential produced by enkephalin are blocked by naloxone. Substance P, on the other hand, has no effect on Ca 2+ action potentials but does reduce the time course of acetylcholine responses in the neurons by a mechanism consistent with enhanced receptor desensitization. Decay of the acetylcholine voltage response in the absence of substance P is described by a single exponential process with a time constant of 4–5s. Coapplication of acetylcholine and substance P results in a second exponential decay process with a time constant of about 1 s that appears after a 200–400 ms lag period. Preincubation with substance P alone does not decrease the peak voltage response or shorten the lag, suggesting that either agonist or activated receptor is necessary for the substance P effect. These findings suggest modulatory roles for the peptides in ganglionic transmission.

Characteristics of acetylcholine-activated channels of innervated and chronically denervated skeletal muscles

Characteristics of the ACh-activated channels before and after denervation of the frog interosseal muscle were studied using the patch clamp technique. Acetylcholine sensitivity was increased on extrajunctional portions of the muscle 7, 42, and 73 days after sectioning of the sciatic nerve. Nonjunctional regions of the innervated muscle appeared to contain one type of ACh channels having a conductance of 28 pS and a mean channel lifetime of 3.8 ms at −90 mV. The denervated muscles contained two classes of channels with conductance of 18 and 28 pS which were present as early as 7 days postdenervation and remained for 93 days. The channel open times of the innervated muscles increased with membrane hyperolarization. The open times of the channels present at 42 days postdenervation showed longer lifetimes than those of innervated muscles and were 10.8 ms and 9.6 ms at −90 mV. These channels also showed less voltage dependence than the control fibers.

Duchenne muscular dystrophy (DMD): Altered acetylcholine (ACh) sensitivity in human myotubes in culture

Duchenne muscular dystrophy (DMD): Altered acetylcholine (ACh) sensitivity in human myotubes in culture

Liposome-delivered phosphatidylcholine enhances the acetylcholine sensitivity of dystrophic mouse myotubes

Myotubes were obtained in vitro from satellite cells of normal and dystrophic C57BL/6J/dydy mice. The acetylcholine sensitivity (mV/nC) of dystrophic myotubes determined with conventional electrophysiological techniques, was lower than that of normal myotubes. Incubation of dystrophic myotubes with liposomes containing phosphatidylcholine (a lipid present in higher amounts in normal adult muscle) significantly increased their acetylcholine sensitivity.

Acetylcholine sensitivity in myotubes of nerve-muscle co-culture cultured with anti-muscle antibodies, α-bungarotoxin and d-tubocurarine

The effects of α-bungarotoxin (α-BuTX). d-tubocurarine (d-TC) and antibodies against muscle extract on acetylcholine (ACh) sensitivity were investigated in developing mouse myotubes in a nerve-muscle co-culture. Antibodies clearly suppressed the ACh potential amplitude in adult mouse diaphragm muscle, but antibodies in muscle pre-treated with d-TC (1 μg/ml) weakly suppressed it. The addition of d-TC to muscle extract dose-dependently inhibited the formation of the immunoprecipitation lines. The exposure of developing myotubes to antibodies for 10 days in culture suppressed both resting potential and ACh potential, whereas co-existence of α-BuTX (1 μg/ml) or d-TC (0.1 mg/ml) with antibodies suppressed ACh potential but did not affect resting potential compared with antibodies alone. The ACh potentials in myotubes cultured with α-BuTX and d-TC alone were also suppressed. The appearance (day 8 in culture) of this suppressive effect by α-BuTX was faster than that (day 11 in culture) of d-TC. These different effects depending on the time in culture may account for the conformational change of developing ACh receptors to α-BuTX and d-TC.

Time course of the induction of acetylcholine receptors in Xenopus oocytes injected with mRNA from Electrophorus electricus electroplax

mRNA from the electroplax of adult Electrophorus electricus was injected into Xenopus oocytes. At various times after injection, the induction of the nicotinic acetylcholine (ACh) receptor in the oocyte membrane was studied electrophysiologically using a two-electrode current clamp. When the ACh sensitivity was induced, membrane potential and conductance rapidly rose from the resting value to their peaks and slowly fell (desensitization) in response to bath-applied 0.1 mM ACh. It took a latency period of 8 ± 2.6 h ( n = 10) from mRNA injection to the first appearance of the ACh sensitivity. During about 10 h incubation after the onset, the increasing speed in the peak of ACh-induced conductance change with incubation time was slow at first and accelerated later. The speed was then decelerated until the induction stages of 60 h. Apparent desensitization properties of the induced receptors changed with an increase of incubation time. During the early induction stages, the conductance decline after its peak followed two exponentials in a minute time region of ACh application: an early slow and a late fast one. The rate of decline in the late fast component slowed down markedly with incubation time. Finally the desensitization followed a single exponential.

Developmentally delayed sensitivity of acetylcholine receptor in myotubes of nerve-muscle cocultures from genetically diabetic mouse embryos.

The neuromuscular junctions of genetically diabetic KK-CAy mice are reported to be hypersensitive to succinylcholine (SuCh) but not to d-tubocurarine (d-TC). Spinal cord-muscle cocultures from normal ddY and diabetic KK-CAy mouse embryos were studied to examine the involvement of genetic factors in this hypersensitivity to SuCh. KK-CAy myotubes were morphologically normal, as determined by light microscopy. KK-CAy myotubes showed a progressive increase in the resting membrane potentials and acetylcholine (ACh) sensitivity with development, but this development was delayed when compared with ddY myotubes. The ACh receptor clusters, fluorescently labeled by fluorescein isothiocyanate conjugated alpha-bungarotoxin (FITC-alpha BuTX), were formed on the surface membrane of KK-CAy myotubes. The developmental increase of the total amount of fluorescence within ACh receptor clusters on KK-CAy myotubes was also slower than that of ddY myotubes. Depolarization by SuCh was sustained at a higher level in KK-CAy myotubes. In regards to the inhibition of ACh potentials, KK-CAy myotubes were not hypersensitive to both SuCh and d-TC when compared with ddY myotubes. These results suggest that the hypersensitivity to SuCh is not dependent on the genetic difference between ddY and KK-CAy mice, and is probably due to the developmentally diabetic state of the neuromuscular junction.

Gamma-Aminobutyric acid receptors on chick ciliary ganglion neurons in vivo and in cell culture

In the chick ciliary ganglion, preganglionic terminals maintain cholinergic synapses on the choroid neurons and both cholinergic and electrical synapses on the ciliary neurons. The preganglionic terminals also contain enkephalin- and substance P-like immunoreactivity, suggesting that transmission through the ganglion is more complicated than is indicated by the known synaptic connections. We report here that embryonic chick ciliary ganglion neurons also have gamma-aminobutyric acid (GABA) receptors and that GABA applied to the ganglion can block transmission elicited by preganglionic stimulation. Studies on the neurons in cell culture indicate that the GABA response is mediated by GABAA receptors: GABA activates a Cl- conductance, and the response can be mimicked by muscimol and blocked by bicuculline or picrotoxin. The GABA receptors are regulated independently from acetylcholine (ACh) receptors on the neurons since the levels of ACh and GABA sensitivity are influenced differently by culture age and by chronic exposure to GABA or elevated K+ concentrations. Application of GABA to intact ciliary ganglia increases the membrane conductance of ganglionic neurons (as in culture), reduces to subthreshold the amplitude of excitatory postsynaptic potentials in the neurons elicited by preganglionic stimulation and completely blocks transmission through the ganglion. A native source of ligand for the receptors in vivo has yet to be identified.

Acetylcholine supersensitivity in the rat heart produced by neonatal sympathectomy.

Effects of neonatal sympathectomy with antiserum to nerve growth factor or 6-hydroxydopamine on the acetylcholine sensitivity of the rat left atria were investigated. Sensitivities to acetylcholine of atria from immunologically and chemically sympathectomized rats were much higher than that of control at 4 weeks of age. These results suggest possible involvement of the sympathetic nervous system in regulation of cardiac cholinergic sensitivity.