Release of [3H]acetylcholine from a modified rat phrenic nerve-hemidiaphragm preparation

Summary Two different preparations of the rat phrenic nerve-hemidiaphragm (whole nerve-muscle preparation, end-plate preparation) were used for studying synthesis and release of radioactive acetylcholine in the absence and presence of cholinesterase inhibitors.When the whole nerve-muscle preparation (110–180 mg) was incubated with [³H]choline, only small amounts of radioactive acetylcholine were synthesized within the tissue. Electrical nerve stimulation of the whole nerve-muscle preparation produced no increase in tritium outflow.Incubation of the end-plate preparation (16–29 mg) which was obtained after removal of most of the muscle mass led to the formation of large amounts of [³H]acetylcholine. Synthesis depended on nerve activity and increased 13-fold during a high loading stimulation (50 Hz), as compared to the synthesis at rest. In a denervated end-plate preparation the formation of [³H]acetylcholine was reduced to 4% of the control preparation. Electrical nerve stimulation of the end-plate preparation produced a release of tritium that could be attributed entirely to the release of [³H]acetylcholine. The stimulated tritium efflux was completely suppressed in a calcium-free medium or in the presence of tetrodotoxin (300 nM). Release could even be detected during a short train of 50 pulses (5 Hz) with a fractional release of about 0.04% of the [³H]acetylcholine tissue content per pulse.It is concluded that the large muscle mass interferes with nerve labelling by a reduction of the [³H]choline supply to the nerve terminals when the whole nerve-muscle preparation is used. Removal of most of the muscle fibres reduces the possibility for [³H]choline to be captured by them and then more radioactive choline can enter the end-plate region. From this end-plate preparation a calcium-dependent release of radioactive transmitter can be measured in the absence of cholinesterase inhibitors.     

Cooperative effects of hemicholinium-3 on high-affinity choline uptake by rat diaphragm

Choline uptake in the end-plate rich area of rat diaphragm is saturable between 0.7 and 16 μM. This choline uptake obeys hyperbolic kinetics and indicates the presence of high-affinity carrier with K_m values of 11.8 and 13.2 μM and V_max values of 37 and 31 nmol choline/h/g tissue respectively. In the presence of hemicholinium-3 the carrier shows sigmoidal kinetics, and hemicholinium-3 is apparently a cooperative effector of choline uptake. The carrier is more calcium than sodium dependent.     

Autoradiography of 14C-choline uptake in endplates and skeletal muscle of mice

Summary The uptake of ¹⁴C-choline into the axonal part of the motor endplate and muscle of mouse diaphragms was investigated by autoradiography. With i. v. doses of 0.1 g/g choline chloride, the uptake into the nerve endings is fast (<2 min) and into muscle slower (>2 min). With higher doses (1.0 g/g) the uptake into muscle tissue is accelerated.The radioactivity in the endplates decreases with a halflife time of 20 min and remains constant in the muscle fibres over 60 min. Denervation by cutting the phrenic nerve reduces the uptake into endplates by 40% within 14 h, but probably induces uptake into regenerating Schwann cells during 30 days. Some compounds with choline-like structure (hemicholinium-3, decamethylen-dicholine, triethyl-choline) reduce the high-affinity uptake of choline into the nerve endings with sublethal doses, whereas tetraethylammonium and N-hydroxyethyl-4-(1-naphthylvinyl)-pyridinium, an inhibitor of cholinacetyltransferase, are less active. Half lethal doses of cocaine, imipramine and reserpine have no significant action on uptake of choline into the endplates. Chlorpromazine slightly diminishes the uptake into endplates. Chlorpromazine and imipramine reduce uptake into the muscle fibres.     

Effects of corticosteroids on neuromuscular blocking actions of d-tubocurarine

Dexamethasone (50 μg/kg) significantly increased the LD₅₀ of d-tubocurarine (d-TC) when administered i.p. simultaneously with d-TC. Choline (50 and 100 mg/kg) gave some protection against the lethal effects of d-TC and the cholinesterase inhibitors neostigmine (250 μg/kg) and physostigmine (1000 μg/kg) provided full protection against doses of d-TC twice the LD₅₀. The blocking effect of d-TC (75 μg/kg) on the sciatic nerve-tibialis anterior muscle preparation was antagonized by dexamethasone. Prednisolone delayed the occurrence of a complete neuromuscular block caused by d-TC in the phrenic nerve-diaphragm preparation, and antagonized the effect of d-TC on short tetanic contractions. d-TC (5 μmol/l) inhibited the [¹⁴C]choline uptake in the endplate-rich region of the rat diaphragm during stimulation. This inhibition was antagonized by dexamethasone as well as by physostigmine. The incorporation of radioactive choline into acetylcholine was inhibited in the presence of d-TC (15 μmol/l), and both dexamethasone and physostigmine counteracted this inhibition. It is concluded from these experiments that d-TC very probably has an effect of the choline carrier system. These experimental results support the hypothesis that glucocoriticoids may improve reduced muscle performance by direct presynaptic effects at the neuromuscular junction.     

Pharmacological actions of some cyclic analogues of choline

Two cyclic choline analogues (3-hydroxy-N,N- dimethylpiperidinium and 2-hydroxymethyl-N,N- dimethylpiperidinium ) and two cyclic homocholine analogues (4-hydroxy-N,N- dimethylpiperidinium and 3-hydroxymethyl-N,N- dimethylpiperidinium ) have been studied with regard to their actions at the cholinergic synapse. All the analogues had some direct depolarizing activity on the frog rectus abdominis muscle but they were less potent in this respect than acetylcholine. Compared to physostigmine, the analogues were weak inhibitors of cholinesterase enzymes. All the analogues were found to have a presynaptic blocking action on the rat phrenic nerve-hemidiaphragm preparation, which was reversed by choline. In addition, they all inhibited the high affinity transport of choline into synaptosomes but only the cyclic choline analogues were found to be acetylated by soluble choline acetyltransferase in vitro. We conclude that the hydroxypiperidinium analogues caused the presynaptic block seen at the neuromuscular junction by inhibiting acetylcholine synthesis.     

Effects of organophosphates on presynaptic events in the vascularly perfused phrenic nerve-hemidiaphragm preparation from the rat

A vascularly perfused phrenic nerve-hemidiaphragm preparation from the rat was developed to study effects of physostigmine and some organophosphate inhibitors on the synthesis and release of endogenous and deuterium-labelled (choline--D9) acetylcholine (ACh) as well as the presynaptic uptake of choline. Choline and ACh were determined by combined gas chromatography/mass spectrometry. Without stimulation the endogenous levels of ACh were 320 pmole/hemidiaphragm for unlabelled and less than 1 pmole/hemidiaphragm of deuterium-labelled ACh. After stimulation at 15 Hz for 1 hr, 460 pmole/hemidiaphragm of unlabelled and 15 pmole/hemidiaphragm of deuterium-labelled ACh were found. Without stimulation the release of unlabelled ACh was 6 pmole/min/hemidiaphragm and for deuterium-labelled 0.2 pmole/min/hemidiaphragm. Evoked release (15 Hz, 1 hr) was 22 pmole/min/hemidiaphragm for unlabelled and 1.8 pmole/min/hemidiaphragm for deuterium labelled ACh. During stimulation and treatment with high concentrations (10(-5)-10(-4) M) of soman, DFP and Vx the level of unlabelled endogenous ACh increased, but the level of deuterium labelled ACh decreased in the diaphragm. During stimulation and treatment with these inhibitors the release of both unlabelled and labelled ACh decreased. During treatment with high concentrations (10(-5)-10(-4) M) of sarin and physostigmine there were no changes in endogenous levels or release of unlabelled or deuterium labelled ACh. The different effects of cholinesterase inhibitors are probably linked to the synthesis and release mechanism of ACh rather than to the choline uptake mechanism.     

The effect of cholinesterase inhibitors and corticosteroids on rat nerve-muscle preparations treated with hemicholinium-3

Corticosteroids improve muscle function impaired by hemicholinium-3 (HC-3). The effects of cholinesterase inhibitors either alone or in combination with the glucocorticoid dexamethasone were studied on nerve-muscle preparations treated with HC-3, both in vivo and in vitro. Administered simultaneously with HC-3 (80 μg/kg by i.p. injection, physostigmine (250–1500 μg/kg) caused a significant, dose-dependent reversal of the neuro-muscular transmission block caused by HC-3 in the sciatic nerve-tibialis anterior muscle preparation of the anaesthetized rat, stimulated at a rate of 10 Hz. The HC-3-blocked neuromuscular transmission was also antagonized by neostigmine (125 and 250 μg/kg). Under the same conditions physostigmine (1000 μg/kg) potentiated the effect of dexamethasone. After i.p. injection of physostigmine (125–1000 μg/kg) and neostigmine (62.5–250 μ/kg) respectively, the LD₅₀ in rats was significantly increased. With physostigmine a sharp maximum was found at 250 μg/kg. Neostigmine, and to a lesser extent physostigmine antagonized the neuromuscular transmission inhibited by HC-3 in the in vitro phrenic nerve—diaphragm preparation of the rat. The HC-3-induced inhibition of both uptake of choline and its incorporation into acetylcholine was antagonized by physostigmine and neostigmine. This antagonism was not altered in the presence of dexamethasone 0.2 μM. The possible implications of these findings, and the mechanism underlying the effects seen with corticosteroids and cholinesterase inhibitors are discussed. It is concluded that under certain circumstances a nerve-muscle preparation treated with HC-3 may be an adequate model for myasthenia gravis.     

Expression and Functional Characterization of Choline Transporter in Human Keratinocytes

Choline is essential for synthesis of the major membrane phospholipid phosphatidylcholine. Moreover, it serves as a precursor for synthesis of the neurotransmitter acetylcholine (ACh). Keratinocytes of the epidermis synthesize and release ACh. The uptake of choline is the rate-limiting step in both ACh synthesis and choline phospholipid metabolism, and it is a prerequisite for keratinocyte proliferation. However, the nature of the choline transport system in keratinocytes is poorly understood. In this study, we examined the molecular and functional characterization of choline uptake into cultured human keratinocytes. Choline uptake into keratinocytes was independent of extracellular Na⁺, saturable, and mediated by a single transport system with an apparent Michaelis-Menten constant of 12.3 μM. Choline uptake was reduced when the keratinocyte membrane potential was depolarized by high K⁺. These results provide evidence that the choline transport activity is potential-sensitive. Various organic cations inhibit the choline transport system. RT-PCR demonstrated that keratinocytes expressed mRNA for choline transporter-like protein 1 (CTL1), mainly the CTL1a subtype. The present biochemical and pharmacological data suggest that CTL1a is functionally expressed in human keratinocytes and is responsible for the uptake of choline and organic cations in these cells.     

Mutual dependence of calcitonin-gene related peptide and acetylcholine release in neuromuscular preparations

To investigate the mutual dependence of calcitonin gene-related peptide (CGRP) and acetylcholine release, we examined the effect of a cholinesterase inhibitor neostigmine on the release of CGRP-like immunoreactivity in rat phrenic nerve-hemidiaphragm muscle preparation, and conversely, the effect of CGRP on [

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]acetylcholine release from motor nerve terminals loaded with [

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]choline in the same preparations of mice. Release of CGRP-like immunoreactivity was increased by electrical nerve stimulation (train of 40 pulses of 200 μs pulse duration and frequency of 50 Hz applied every 10 s) in the whole preparation but not in the segmental preparation containing the endplate region. Neostigmine (0.1–0.3 μM) enhanced the resting release of CGRP-like immunoreactivity in a concentration-dependent manner, whereas it depressed the nerve-evoked release of CGRP-like immunoreactivity. CGRP (1 μM) added to perfusate decreased nerve-evoked [

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]acetylcholine release. These results suggest that CGRP, which is released by electrical nerve stimulation or a cholinesterase inhibitor in intact skeletal muscles, negatively modulates nerve-evoked acetylcholine release.     

Dendrotoxin-like effects of noxiustoxin.

Noxiustoxin from the Mexican scorpion (Centruroides noxius Hoffmann) is known to block neuronal K+ channels. Noxiustoxin facilitated acetylcholine release in chick biventer cervicis nerve-muscle preparations, but not in mouse phrenic nerve-hemidiaphragm preparations. Noxiustoxin displaced binding of a radiolabelled dendrotoxin from synaptosomal membranes from rat brain, with a Ki of 10(-10) M. It is concluded that noxiustoxin shares some pharmacological properties with the K+ channel blocking dendrotoxins.     

The action of papaverine on antidromic activity in isolated nerve-muscle preparation of the rat

The actions of papaverine and of (+)-tubocurarine were compared in isolated nerve muscle preparation of the rat. An antidromic discharge in the phrenic nerve-hemidiaphragm preparation was elicited by electrical stimulation of the phrenic nerve after neostigmine administration. Spontaneous antidromic discharge was seen in Krebs solution containing half the usual concentration of calcium and neostigmine. Papaverine completely blocked both the response evoked by electric stimulation and the spontaneous discharge, whereas (+)-tubocurarine blocked the evoked response but only slightly inhibited the spontaneous antidromic firing in the media with half calcium concentration. The mechanism of antinicotinic action of papaverine and (+)-tubocurarine seems to differ.     

Effect of cellular desialylation on choline high affinity uptake and ecto-acetylcholinesterase activity of cholinergic neuroblasts.

It is not definitely established whether choline can be synthesized by nerve tissue (1,2). Its uptake by the neuronal membrane may be an important regulator of neuronal phosphatidylcholine and acetylcholine metabolism. An energy-dependent component of the choline uptake system has recently been found in neuroblastoma cell cultures (3). Choline uptake in cholinergic neuroblasts is markedly reduced by inhibition of cholinesterase activity (4). Further information about the system for choline transport still is lacking.To gain insight into the biochemical mechanism(s) at the cell surface which are involved in choline uptake by nerve cells, cultured cholinergic neuroblasts in the present study were subjected to gentle sialidase treatment, and choline uptake and acetylcholinesterase activities (AChE, EC 3.1.1.7) were determined in parallel. Removal of a portion of the cell surface sialic acid markedly reduced choline uptake and concurrently enhanced AChE activity. These findings suggest an interrelatedness of choline uptake and acetylcholinesterase activity mediated by sialic acid components in the outer surface of the cell.     

Interaction of choline with muscarine receptor-stimulated phosphoinositide metabolism in the rat brain

Summary Previous receptor binding studies had shown that choline can interact with low potency with muscarine cholinoceptors. In the present study we have investigated whether choline is capable of functionally activating muscarine receptors by investigating its ability in stimulating the hydrolysis of phosphoinositides, a response believed to be coupled in brain to the M1 subtype of muscarine receptors. The results indicated that choline was only a very weak inducer of inositol phosphates (InsPs) accumulation in rat cerebral cortex slices as compared with acetylcholine or charbachol. Maximal increase of InsPs accumulation, at a choline concentration of 10 mM, was only 39±7%, as compared with the 4- to 6-fold stimulation induced by the other compounds. This effect of choline was not modified by physostigmine nor by the uptake inhibitor hemicholinium-3. At high concentrations, however, choline antagonized the stimulatory effect of acetylcholine and carbachol, suggesting that it might act as a partial agonist at this subtype of muscarine receptors, similar to what has been observed with oxotremorine. Choline had no effect on noradrenalinestimulated InsPs accumulation.     

Actions of delta9-tetrahydrocannabinol on neuromuscular transmission in the rat diaphragm.

The actions of in vivo and in vitro administration of Δ⁹-tetrahydrocannabinol on neuromuscular transmission were studied using the rat phrenic nerve-diaphragm preparation. Diaphragms obtained from animals treated acutely with intravenously administered Δ⁹-tetrahydrocannabinol (4 $\text{mg}\text{kg}$) showed an increase in both frequency and amplitude of miniature end-plate potentials as well as a prolonged duration of the end-plate potentials. Similar changes were observed when diaphragms were treated in vitro with 10^?4 M Δ⁹-tetrahydrocannabinol. Δ⁹-Tetrahydrocannabinol administered either in vivo or in vitro had no significant effect on cholinesterase activity, and had no effect on the resting membrane potential of muscle fibres. [¹⁴C]-Δ⁹-Tetrahydrocannabinol was not specifically localized at the end-plate regions but was distributed equally throughout the muscle fibres. While the data suggest that Δ⁹-tetrahydrocannabinol acts mainly on the presynaptic site, an additional action on the postsynaptic membrane cannot be excluded.     

Biochemical and pharmacological studies on the sulfonium analogues of choline and acetylcholine

Sulfocholine was found to be a substrate for choline acetyltransferase and acetylsulfocholine was a substrate for acetylcholinesterase and cholinesterase. Sulfocholine, like choline, was taken up by an active transport mechanism into brain slices. When ¹⁴C-labelled sulfocholine was injected into mice, formation of labelled acetylsulfocholine in the brain was demonstrated. The equipotent cholinomimetic molar ratio of acetylcholine and acetylsulfocholine on the tibialis anterior preparation of the cat was 6.6 : 1. The response elicited by sulfocholine on the stimulated tibialis anterior preparation was similar to that of choline, and was antagonized by D-tubocurarine. Sulfocholine had a restorinng action on the hemicholinium-inhibited tibialis anterior preparation, although weaker than that of choline. It is concluded, that the action of sulfocholine on neuromuscular transmission consists mainly of a stimulating effect on the motor end-plate, but a presynaptic action, leading to an increased output of acetylcholine, is also suggested.     

On the ability of choline and its analogues to interact with muscarinic cholinergic receptors in the rat brain.

Choline displaced [3H]QNB binding from rat brain muscarinic receptors competitively, (Ki = 460 microM) but it was only 1/1000th as potent as ACh. Deanol was an extremely weak displacer of [3H]QNB binding while hemicholinium-3 was 50 times more potent than choline. Although brain levels of choline are well below its Ki value for muscarinic receptors, choline may directly interact with rat brain muscarinic receptors in some circumstances.     

Pharmacological actions of HS-6, an oxime, on the neuromuscular junction.

The effect of HS-6 [1-(2-hydroxyiminomethylpyridinium)-1-(3-carboxamidopyridinium)-dimethyl ether] on neuromuscular (NM) transmission was investigated using chick biventer cervicis (CBC), rat diaphragm (RD) and guinea pig ileum longitudinal muscle strip (GPLS) preparations. In the CBC preparation HS-6 did not cause a contraction, whereas it produced a dose-related contraction of the GPLS preparation. HS-6 produced a hemicholinium-like NM blockade in the CBC and RD preparations. This was supported by the fact that HS-6 inhibited choline uptake in erythrocytes. HS-6 inhibited the contractions of various nicotinic agonists in the CBC preparation and augmented the acetycholine contractions. The latter was due to AChE inhibition produced by HS-6.     

The local anaesthetic activity of a benzotriazinium salt.

1 The local anaesthetic properties of 2-n-propyl-4-p-tolylamino-1,2,3-benzotriazinium iodide (TnPBI) were compared with those of lignocaine hydrochloride on intact and desheathed sciatic nerves of the frog, on the phrenic nerve-hemidiaphragm preparation of the rat, and by the intradermal wheal test in the guinea-pig. 2 Both TnPBI and lignocaine were more potent on desheathed than on intact sciatic nerves. The potency of TnPBI was affected more than that of lignocaine by the presence of the sheath in intact nerves. 3 Both drugs inhibited conduction in the rat phrenic nerve, as shown by the reduction in twitch tension of the diaphragm elicited by nerve stimulation. TnPBI also caused an initial augmentation of the twitch tension of the diaphragm when applied directly to the muscle. 4 TnPBI was shown to be approximately twice as potent as lignocaine by the guinea-pig intradermal wheal test. 5 These results are discussed in view of the known effects of TnPBI on intracellular calcium storage.     

The effect of some foreign anions on suxamethonium blockade of the isolated rat diaphragm preparation

1. The pharmacology of suxamethonium blockade of the rat phrenic nerve-diaphragm preparation has been studied in the presence of some foreign anions.2. Blockade proceeded in two distinct phases in both normal and modified Krebs solution.3. In normal Krebs solution the characteristics bore no close resemblance to either depolarizing or competitive type blockade.4. In the presence of foreign anions the characteristics of the blockade more closely resembled those of depolarization.5. There was an increase in the sensitivity of the motor end-plate region of the muscle to the depolarizing action of acetylcholine, carbachol and suxamethonium in the presence of the anions.6. Although the anions enhanced the depolarizing activity of suxamethonium, the blocking potency of the drug was unaltered.7. It is suggested that end-plate depolarization plays little part in suxamethonium blockade of the isolated rat diaphragm and that desensitization is the primary cause of the blockade.     

The neuromuscular action of Ancylometes sp. spider venom in the rat phrenic nerve-diaphragm preparation.

The effects of Ancylometes sp. venom on muscle contraction and bioelectrical potentials were investigated in the rat phrenic nerve diaphragm muscle preparation. The venom (50 microg/ml) depolarized the diaphragm muscle fiber membranes. This effect was abolished by tetrodotoxin and by reduction of the sodium concentration of the Tyrode solution. The increase in the frequency of miniature end plate potentials induced by the venom was also suppressed by tetrodotoxin (3 microM). These results indicate that the venom may activate voltage-dependent sodium channels in cell membranes. All of the effects of Ancylometes sp. venom on this nerve muscle preparation (i.e. increase in twitch tension, spontaneous small phasic contractions and increase in the frequency of miniature end-plate potentials) may be explained in terms of its action on sodium channels.