Robin D. Veldsema-Currie

The effect of corticosteroids and hemicholinium-3 on choline uptake and incorporation into acetylcholine in rat diaphragm

The glucocorticoids prednisolone and dexamethasone antagonize the inhibition by hemicholinium-3 of both the rate of choline uptake and the incorporation of choline into acetylcholine in the rat diaphragm. Aldosterone has no such effects. It is concluded that the beneficial effect of glucocorticoids in the treatment of myasthenia gravis may be due not only to immunosuppression, but also to some direct effect on presynaptic events perhaps via a choline carrier or an enzyme of choline metabolism.

Effects of corticosteroids on neuromuscular blocking actions of d-tubocurarine

Dexamethasone (50 microgram/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 microgram/kg) and physostigmine (1000 microgram/kg) provided full protection against doses of d-TC twice the LD50. The blocking effect of d-TC (75 microgram/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 mumol/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 mumol/l), and both dexamethasone and physostigmine counteracted this inhibition. It is concluded from these experiments that d-TC very probably has an effect on the choline carrier system. These experimental results support the hypothesis that glucocorticoids may improve reduced muscle performance by direct presynaptic effects at the neuromuscular junction.

Presynaptic, facilitatory effects of the corticosteroid dexamethasone in rat diaphragm: Modulation by beta-bungarotoxin

Low concentrations of dexamethasone (up to 200 nM) increase the accumulation of choline (Ch) and its incorporation into acetylcholine (ACh) in the endplate rich area (EPA) of stimulated and unstimulated diaphragms in the presence of 10 microM Ch. Tissue ACh is not significantly altered, even after 140 min incubation. The specific radioactivity of the ACh in the EPA is thus increased by dexamethasone (Dex). The corticosteroid has no effects on acetylcholinesterase or choline acetyltransferase in diaphragm extracts. In the same medium, the amplitudes of the MEPPs, MEPCs and EPCs are also increased by Dex. Neither the quantal content of the EPCs nor the MEPP frequency, nor the half decay time of the MEPCs are altered. Therefore Dex (200 nM) increases both the resting and evoked output, and turnover of ACh in rat diaphragm. Beta-bungarotoxin (beta-BuTx) antagonizes the Dex-induced increase in Ch accumulation and its incorporation into ACh, and abolishes the increases in MEPC- and EPC-amplitudes, providing further argument for a presynaptic effect of Dex. In continuously-stimulated diaphragms, beta-BuTx causes an accumulation of ACh which is much greater than in unstimulated tissue. This accumulation of ACh is less in the presence of Dex, provided that Dex is added before beta-BuTx. The interaction of Dex and beta-BuTx is discussed in terms of their possible presynaptic sites of action.

Dexamethasone affects radioactive choline uptake in rat diaphragm nerve endings and not in muscle fibres

The initial rate of radioactive choline (Ch) uptake in the endplate-rich area (EPA) of both stimulated and unstimulated hemidiaphragms is significantly increased by 0.2 microM dexamethasone (Dex) in the presence of 10 microM Ch. In autoradiographs, the mean grain densities above the muscle fibres are not altered by Dex. The mean grain densities above the nerve endings are significantly increased in the presence of Dex in stimulated tissue, and slightly but not significantly increased in unstimulated tissue. There is a positive correlation between the initial rate of Ch uptake in the EPA and the amount of isotope in the nerve terminals, in the absence and presence of Dex. Without correcting for the large amount of diffusion which occurs, the ratio of the grain densities above the nerve terminals to that above the muscle fibres in the presence of Dex is 2.12 in stimulated tissue, and 1.40 in unstimulated tissue. The ratio in the stimulated tissue is significantly greater than the control ratio in the absence of Dex (1.66). Therefore, Dex affects radioactive Ch uptake in nerve endings and not in muscle fibres in the rat diaphragm. The stimulation-induced increase in the uptake of isotope into the nerve endings is abolished in a Na+-depleted medium, and in the absence of Ca2+. Dex has no effect on this abolition. We conclude that relatively low concentrations of Dex affect Ch transport in rat diaphragm nerve endings by a mechanism as yet to be defined.

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.

Presynaptic, high-affinity glutamate uptake in excitatory neuromuscular junctions of the retractor unguis muscle of the locust: determination of the Km value using high resolution autoradiography

SummaryUsing high resolution autoradiography the Km of the presynaptic, Na+-dependent and aspartate inhibited, high affinity glutamate uptake in insect neuromuscular junctions was determined. The values found (approx. 15 μM) did not differ for the glia and terminal axons. This Km value agrees with those values determined by classical biochemical techniques for presynaptic high affinity uptake systems in insect neuromuscular junctions and glutamatergic central nervous systems.

Acetylcholine and an unidentified, muscle-contracting factor in the venom of the bumblebee, Bombus terrestris L.

Extracts of venom reservoirs as well as dilutions of venom droplets collected from the tip of the sting of Bombus terrestris were tested for biological activity on the isolated guinea-pig ileum, the frog rectus abdominis, and the rat phrenic nerve-hemidiaphragm preparation. The venom contains a cholinergic factor, apparently identical to acetylcholine, at a concentration of about 30 micrograms per venom reservoir. This was confirmed using radioimmunoassay of acetylcholine. The venom also contains a component causing a slow contraction of guinea-pig ileum and rat diaphragm. This component is heat stable, non dialysable, and is slowly destroyed in the venom preparation at about 0 degrees C.

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 micron. This choline uptake obeys hyperbolic kinetics and indicates the presence of high-affinity carrier with Km values of 11.8 and 13.2 micron and Vmax 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.

Acetylcholine stores in rat diaphragm are increased by higher concentrations of dexamethasone

In indirectly stimulated hemidiaphragms, dexamethasone (Dex, 2 microM) causes a significant increase in tissue acetylcholine (ACh) content. No increase in tissue ACh is found with 0.2, 0.6 or 6 microM Dex. Physostigmine (Physo, 15 microM) also causes an increase in tissue ACh, which is even greater in the presence of Dex (6-25 microM). There is no increase in ACh due to Dex, with 50 microM Dex plus Physo. The order of addition is important, as the Dex-induced increase in ACh is only found when Dex is added before Physo. No increase in twitch tension is found with any of these treatments. No Dex-induced increase in ACh is found with unstimulated hemidiaphragms. Similar increases in ACh are also found with other glucocorticoids (plus Physo), namely prednisolone (6-9 microM), corticosterone (2 microM) and hydrocortisone (2 microM). The mineralocorticoid aldosterone (2 microM), and other types of steroids cause no increase in tissue ACh. The increases in hemidiaphragm ACh are not found in a Na+-depleted medium, or in a medium containing 20 mM Mg2+ extra. The increase in ACh due to Physo is Ca2+-dependent, even though an increase in ACh due to Dex plus Physo is found in the absence of Ca2+. No increase in ACh due to either Physo, or Dex plus Physo are found in the presence of the nicotinic antagonists (+)-tubocurarine (5 microM) or alpha-cobrotoxin (5 micrograms/ml), while the muscarinic ligands atropine or oxotremorine (10 microM) abolish the extra increase in ACh due to Dex.(ABSTRACT TRUNCATED AT 250 WORDS)

Dexamethasone reduces the high-affinity binding of hemicholinium-3 to rat phrenic nerve endings in vitro: an autoradiographic study

Receptor autoradiography has revealed high-affinity binding sites for hemicholinium-3 (HC-3) in the phrenic nerve endings of rat diaphragm. It has been demonstrated that 200 nM dexamethasone (Dex) in vitro reduces the binding of HC-3 to these high-affinity sites. It seems likely that the decrease in inhibitory effects of HC-3 on choline uptake and acetylcholine synthesis in rat diaphragm caused by Dex, should be the result of an interference of Dex with the binding of HC-3 to its high affinity sites.