Pyridostigmine kinetics with and without renal function.

Pyridostigmine kinetics were examined under conditions of clinical use as an antagonist of nondepolarizing neuromuscular blockade in anesthetized patients with and without renal function. Pyridostigmine serum levels were assayed by gas-liquid chromatography, and data were fitted to a 2-compartment kinetic model. Pyridostigmine kinetics following renal transplantation (n = 5) were not different from those in patients with normal renal function. Renal function (n = 5) elimination half-life increased from 112 +/- 12 min (mean +/- SD) to 379 +/- 162 min, and serum clearance decreased from 9 +/- 2 ml/kg/min to 2 +/- 0.6 ml/kg/min in anephric patients (n = 4). We conclude that renal function accounts for 75% of pyridostigmine clearance.

Effect of prednisolone on neuromuscular blocking in mice in vivo.

The working capacity of inbred female Swiss mice on the rotating rod was tested in an investigation of the effect of previously injected prednisolone, 1.2 mg, on the neuromuscular blocking effect of two different doses of d-tubocurarine and of low-dose hemicholinium-3. The effect of both drugs could be partially or wholly antagonized by prednisolone. The consequences of this finding are discussed in relation to the beneficial effect of prednisone in patients with myasthenia gravis.

Effect of corticosteroids on sciatic nerve-tibialis anterior muscle of rats treated with hemicholinium-3. An experimental approach to a possible mechanism of action of corticosteroids in myasthenia gravis.

We studied the effect of intraperitoneally administered corticosteroids on the neuromuscular transmission in the sciatic nerve-tibialis anterior muscle preparation of the anesthetized rat stimulated at a rate of 10 Hz. Administered simultaneously with hemicholinium-3 (HC-3), 80 mug per kilogram (that is, half the lethal dose for 50 percent survival), prednisolone and dexamethasone cause a marked reversal of the block of the neuromuscular transmission caused by HC-3. The effect of aldosterone is very small. The blocking action of d-tubocurarine is not antagonized by either prednisolone or dexamethasone. Choline provides total protection against the HC-3 blockade, whereas physostigmine, in a just sublethal dose, is ineffective. We tentatively conclude that in myasthenia gravis the carrier-mediated transport of choline into the nerve endings may be deficient and that the beneficial effect of corticosteroids in this condition is based on their ability to ameliorate the deficient choline transport.

Ligated renal pedicles and duration of action of neostigmine and pyridostigmine.

1. The duration of neostigmine or pyridostigmine antagonism of a (+)-tubocurarine (Tc) neuromuscular blockade was determined in the cat anterior tibialis-peroneal nerve preparation with and without ligated renal pedicles. 2. The infusion rate of Tc required to maintain a 90% depression of twitch tension was reduced from 8.8 +/- 1.4 (s.e.) to 3.4 +/- 0.6 mugkg-1 min-1 by renal pedicle ligation. 3. Renal pedicle ligation resulted in an increased duration of antagonism of Tc by both neostigmine and pyridostigmine.

Some effects of dimethyl sulphoxide (DMSO) on the frog neuromuscular junction.

1. Dimethyl sulphoxide (DMSO) partially reversed neuromuscular blockade brought about by the action of (+)-tubocurarine or Mg(2+) on the frog sartorius nerve-muscle preparation.2. The amplitude and duration of the endplate potential (e.p.p.) were increased by DMSO at concentrations of 70 mM or greater.3. Miniature endplate potentials were raised in frequency, prolonged in duration and increased in amplitude by DMSO at concentrations of 141 mM or greater, but the increase in amplitude was generally less than in the case of the e.p.p.4. The resting muscle membrane potential was significantly depolarized by DMSO at 70 mM or greater concentrations, both at the endplate and remote from an endplate.5. The reversal of neurmuscular blockade by DMSO can be explained in terms of its previously reported ability to inhibit cholinesterase activity, together with the depolarizing action on muscle.

Interaction of competitive antagonists: the anti-curare action of hexamethonium and other antagonists at the skeletal neuromuscular junction.

1. In the rat isolated diaphragm preparation hexamethonium and other low potency competitive antagonists of acetylcholine (ACh), including gallamine and hyoscine butylbromide, reverse block by the potent antagonists tubocurarine, pancuronium and alcuronium. 2. In the presence of tubocurarine, hexamethonium increases the amplitude of the end-plate potential without increasing the quantal content. It enhances the response to ACh applied iontophoretically to the end-plate but does not enhance the response to ACh applied in the bath. 3. The anti-curare effect of hexamethonium is abolished in the diaphragm of the rat, guinea-pig and mouse by inhibitors of acetylcholinesterase. The effect is not observed in the indirectly stimulated toad sartorius muscle. 4. The effect is explained if tubocurarine does not dissociate appreciably in the time taken for ACh to achieve high occupancy of receptors, so that a fraction of receptors is completely excluded from occupation by ACh. Equilibration with hexamethonium reduces the fraction excluded by tubocurarine and the transmitter now competes with hexamethonium for more receptors and produces a larger response. 5. On the basis of this explanation the half-time for dissociation of tubocurarine must be about 1 millisecond. It follows that tubocurarine does not act competitively with ACh at synapses when transmitter action is sufficiently brief, and that its binding to the receptor is probably diffusion-limited.

The interaction between hexamethonium and tubocurarine on the rat neuromuscular junction.

The ability of hexamethonium (C6) to reverse the neuromuscular blocking action of tubocurarine (Tc) has been reinvestigated at the voltage clamped endplate of the omohyoid muscle of rat. The possibility that a weak anticholinesterase action of C6 could contribute to the paradoxical potentiation of the peak amplitude of the endplate response has been examined. C6 (50-200 microM) caused an increase in the amplitude of nerve-evoked endplate currents (e.p.cs) recorded in the presence of 0.6 microM Tc. The effect decreased with hyperpolarization of the muscle fibre. Irreversible inhibition of acetylcholinesterase resulted in a loss of the anti-curare effect of C6. C6 did not cause an increase in e.p.c. amplitude when acetylcholine (ACh) receptors were blocked irreversibly by alpha-bungaratoxin. When transmission was blocked by increased Mg2+ concentration, C6 (50-400 microM) reduced the amplitude of e.p.cs without appreciably affecting their time course. C6 caused a decrease in the amplitude of miniature endplate currents (m.e.p.cs) the effect being slightly increased when the fibre was hyperpolarized. An e-fold increase in the effectiveness of C6 occurred with approximately 58 mV hyperpolarization. High concentrations (greater than 400 microM) affected the time course of m.e.p.cs in a manner suggestive of open channel block, but this was not evident at 200 microM, the concentration that was most effective in reversing Tc block. When tested against responses to short ionophoretic pulses of agonists, C6 was less effective against ACh (EC50ca. 300 microM) than against carbachol (CCh) (EC50 100 microM). When cholinesterase was irreversibly inhibited, C6 blocked responses to both agonists equally (EC50ca. 100 microM). The effectiveness of C6 in blocking the action of CCh was reduced 10 fold in the presence of 0.6 microM Tc, implying that the two antagonists compete for the same binding site. C6 (50-200 microM) in the presence of Tc (0.6 microM) increased the response to ionophoretically applied ACh but not that to CCh. C6 was equipotent in blocking m.e.p.cs and responses to ionophoretically applied ACh whereas Tc was more potent against the exogenously applied agonist. C6 was a weak inhibitor of acetylcholinesterase activity in rat muscle homogenates (EC50 1.5 mM). The results are discussed in terms of the kinetic hypothesis advanced by Ginsborg & Stephenson (1974) to account for the Tc reversal phenomenon. It is concluded that this theory can explain most of the effect on e.p.cs, but that the weak anticholinesterase action of C6 is also a factor, particularly in the reversal of Tc block of ionophoretic responses.

Intensive anti-oxidant pretreatment retards motor nerve degeneration.

Intensive pretreatment of cats with a combination of the antioxidants D-alpha-tocopherol (200 IU) and selenium (50 micrograms) once daily for 5 days (p.o.) was found to significantly preserve the functional capacity of degenerating soleus motor nerve terminals (measured at 48 h after axon section at the hip) in the in vivo soleus nerve-muscle preparation. The preservation of function was apparent in terms of: a greater soleus contractile response to nerve stimulation at low frequencies, a more rapid recovery from D-tubocurarine-induced neuromuscular block, and a better maintenance of tetanic contractile tension during high-frequency nerve stimulation. The ability of antioxidants to retard the anterograde axonal degeneration (i.e. 'Wallerian') process suggests that lipid peroxidation may be a fundamental mechanism of neuronal degeneration.

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.

Anticholinesterase activity and structure activity relationships of a new series of hemicholinium-3 analogs.

Piperidine derivatives of hemicholinium-3 were synthesized and included the following spacing groups between the bis-cationic heads: trans,trans-bicyclohexyl, phenanthrene, naphthalene and biphenyl. Anticholinesterase activity was determined using rat striatal synaptosomal cholinesterase, bovine erythrocyte acetylcholinesterase and horse serum butyrylcholinesterase. Hemicholinium-3 has little anticholinesterase activity but when the choline moiety of hemicholinium-3 is replaced with selected heterocyclic amine ring systems active inhibitors can be obtained. Results in this communication identify several quaternary amines which are equipotent with physostigmine for inhibition of cholinesterase. Several tertiary amines were also found to be active. Optimal anticholinesterase activity of these piperidine derivatives appear to be related to the necessity of 14 A interatomic distance between the cationic heads as well as C = O substitution in the phenylethyl spacing moiety. Reduction of C = O to secondary alcohol or CH2 results in decreased activity. The anticholinesterase activity is not only related to internitrogen distance and C = O substitution but also structural planarity and positional isomerism of the quaternary cationic head.

Neuromuscular blocking action of cadmium and manganese in isolated frog striated muscles.

In isolated frog sciatic nerve--gastrocnemius muscle preparations, exposure for 60 min to Cd2+ (0.1-2 mM) caused a marked attenuation of the twitch tension developed by stimulation of nerves but no or only a slight inhibition of the tension produced by direct stimulation at maximum twitch height. The inhibitory effect was partially reversed by 1 mM cysteine. In frog sartorius muscles, the addition of Cd2+ (0.1 mM) alos abolished the end-plate potential evoked by nerve stimulation but did not suppress potential changes induced by iontophoretically applied acetylcholine. The addition of Mn2+ (2 and 5 mM) attenuated the response of muscles to both direct and indirect stimulation; a greater attenuation of the latter was observed. The inhibition was not reserved by cysteine but was partially reversed by excess Ca2+. Contractile responses of frog rectus abdominis muscles to acetylcholine were not significantly affected by Cd2+ and Mn2+ but were attenuated by d-tubocurarine in a dose-dependent manner. Impulse conduction along sciatic nerves was not impaired by Cd2+ and Mn2+ but was by procaine. It appears that Cd2+ interferes with the release of acetylcholine from motor nerve terminals by reducing the transmembrane influxes of Ca2+, the influx possibly relating to SH groups of membrane constituents.

The actions of three diaminopyridines on the chick biventer cervicis muscle.

The effects of 2,3-, 2,6- and 3,4-diaminopyridine were investigated on the isolated chick biventer cervicis muscle preparation. All three compounds reversed tubocurarine blockade and augmented twitch height in indirectly stimulated preparations. Less twitch augmentation was observed in directly stimulated preparations. 3,4-Diaminopyridine was the most effective of the compounds in facilitating neuromuscular transmission, but exhibited less convulsant activity than 4-aminopyridine. 3,4- and 2,3-diaminopyridine in high concentrations caused contractures that were inhibited by erabutoxin b or by beta-bungarotoxin. It is suggested that the diaminopyridines increase both evoked and spontaneous acetylcholine release.

The prejunctional inhibitory effect of suramin on neuromuscular transmission in vitro.

The P2 purinoceptor antagonist suramin reverses skeletal muscle paralysis evoked by non-depolarizing neuromuscular blocking agents in vitro and in vivo. To further study the action of suramin on neuromuscular transmission, (miniature) endplate potentials ((m.)e.p.ps), motor nerve terminal currents and the release of radiolabeled acetylcholine was measured in isolated nerve-muscle preparations. In preparations paralysed by low Ca2+/high Mg2+ conditions, suramin (10 microM-1 mM) induced a concentration-dependent decrease in quantal content of the e.p.ps without affecting m.e.p.ps. Suramin reversed neuromuscular block by d-tubocurarine in these preparations. In erabutoxin paralysed preparations, suramin (40 microM-1 mM) inhibited the motor nerve terminal currents related to Ca2+ influx concentration-dependently, but did not affect Na+ currents. Suramin-induced inhibition of Ca2+ currents was not antagonized by ATP gamma S. Suramin (300 microM) reduced [14C]acetylcholine outflow in non-paralysed rat phrenic nerve-hemidiaphragm preparations by 32%. As suramin did not chelate Ca2+, these results indicate that suramin inhibits neuromuscular transmission by blocking prejunctional Ca2+ channels, thereby decreasing acetylcholine release upon nerve stimulation.

The actions of aminopyridines on avian muscle.

The effects of 2-, 3-, and 4-aminopyridines were investigated on the isolated chick biventer cervicis nerve-muscle preparation and on nerve-free cell cultures of embryonic chick skeletal muscle. All 3 compounds reversed tubocurarine blockade and augmented twitch height in indirectly stimulated biventer cervicis preparations. 4-Aminopyridine was approximately 10 times more potent than 2-, or 3-aminopyridine. Twitch augmentation was also seen in directly stimulated preparations but to a much lesser extent. The compounds did not have significant anticholinesterase activity, nor did they have any depolarizing activity when tested on nerve-free cultured muscle fibres. At high concentrations the aminopyridines produced a maintained contracture in the biventer preparations which was enhanced by neostigmine and inhibited by tubocurarine. It is suggested that the aminopyridines facilitate neuromuscular transmission by increasing acetylcholine release in response to nerve stimulation, and that the compounds can also increase spontaneous transmitter release.

The pharmacology of anatoxin-a(s), a neurotoxin produced by the freshwater cyanobacterium Anabaena flos-aquae NRC 525-17.

Anatoxin-a(s) [antx-a(s)] is produced by Anabaena flos-aquae clone NRC 525-17 and is different from anatoxin-a, a known depolarizing agent produced by A. flos-aquae NRC 44-1. Purification of antx-a(s) from lyophilized cells involved extraction with 1.0 M acetic acid: ethanol (80:20), column chromatography (Sephadex G-15 and CM-Sephadex C-25) and high performance liquid chromatography. Purified toxin has an LD50 (i.p., mouse) of approximately 50 micrograms/kg. Gross pharmacological tests of antx-a(s) on isolated chick biventer cervicis and frog rectus abdominis muscles showed no direct agonistic effect. Instead, antx-a(s) augments the acetylcholine response and antagonizes the actions of d-tubocurarine. Twitch potentiation and tetanic fade were observed on isolated rat phrenic nerve--diaphragm muscle when stimulated indirectly at different frequencies. In acute toxicity tests with mice and rats the signs of poisoning were indicative of excessive cholinergic stimulation. Mice pretreated with atropine sulfate showed longer survival times and no parasympathomimetic signs of toxicity. The mice still died of respiratory arrest with convulsions, which indicated that toxicity is due to more than just the peripheral muscarinic action of antx-a(s). Assays of serum cholinesterase of rats in acute toxicity tests showed complete inactivation of the enzyme at doses of 350 and 600 micrograms/kg. It was concluded that antx-a(s) may be acting as an anticholinesterase, thereby causing toxicity.

Anti-curare effect of plasma from patients with thermal injury.

Following severe thermal injury, patients are resistant to non-depolarizing muscle relaxants. Although this resistance has been well documented clinically, little is known about its etiology. We have tested the hypothesis that circulating factors contribute to the decreased potency of neuromuscular blockers following burns. The potencies of d-tubocurarine (2 microM) or pancuronium (2 microM) dissolved in plasma from either burned or control human subjects were tested on the indirectly stimulated (0.2 Hz) rat phrenic nerve-hemidiaphragm preparation. The muscle relaxants produced less neuromuscular blockade when dissolved in plasma from burned patients than when they were dissolved in plasma from controls. Thus, circulating factors are involved in the decreased potency of non-depolarizing neuromuscular blocking drugs.

A comparison of the antagonisms by neostigmine and diaminopyridine against the neuromuscular block caused by cobrotoxin and (+)-tubocurarine.

Cobrotoxin was about 11-fold more potent than (+)-tubocurarine on a weight basis in blocking neuromuscular transmission in mouse isolated phrenic nerve-diaphragm preparations. Neostigmine and diaminopyridine increased the concentrations of cobrotoxin for 70% inhibition of indirect contraction by 290 and 320%, and increased those of (+)-tubocurarine by 180 and 230%, respectively. More than additive increases were obtained when neostigmine and diaminopyridine were used simultaneously. Cobrotoxin, however, was only 6-fold more toxic than (+)-tubocurarine after intraperitoneal injection in mice. The lethal dose of (+)-tubocurarine was increased by 80% when both antidotes were used together, but only by 15-20% when used alone. In contrast, the lethality of cobrotoxin was not decreased by these drugs. Unexpectedly, the time to death after treatment with cobrotoxin was shortened when mice were pretreated with these antidotes.

Study of in vitro and in vitro effects of isradipine in skeletal muscles and interaction with some drugs.

The effects of the calcium channel blocker isradipine were studied in the indirectly and directly stimulated mouse diaphragm and in the anesthetized rat to determine its potency, reversibility and interaction with a number of drugs. Initially, it potentiated both indirect and direct twitches followed by a reduction. With tetanic contractions, no potentiation was obtained, only a reduction, which was complete or near complete at the highest concentration tested (10(-4) M). In combination, isradipine reduced the IC50 and IC90 values for the antibiotics gentamicin, polymyxin B and clindamycin, d-rubocurarine and magnesium ions. Depression of contraction caused by isradipine or in combination could be reversed to varying degrees by washout, elevated calcium ions, neostigmine or 4-aminopyridine. Spontaneous recovery from the effects of isradipine alone or in combination was slow and usually incomplete. For in vivo experiments, severe cardiovascular depressant effects of isradipine limited its exposure to lower concentrations and for shorter periods. Under these conditions, it had no effect on heart rate. However, both systolic and diastolic blood pressure were significantly reduced, while pulse pressure was increased. After an initial potentiation muscle contraction was maximally reduced to 55% of control. This study indicates that acute administration of isradipine may aggravate neuromuscular effects of antibiotics, muscle relaxants or hypermagnesemia, although it is unlikely that spontaneous recovery or reversibility of muscular activity by suitable reversal agents will be affected. However, prolonged use of the drug may be more difficult to reverse.

The potency ratio of galanthamine and neostigmine on the reversal of the tubocurarine block in the isolated rat diaphragm.

The potency ratio of galanthamine and neostigmine was studied on the rat phrenic nerve-diaphragm preparation. This ratio appeared to be about 18 on a weight base. The potency ratio in man in vivo, is about 20. Hence we concluded that this potency ratio might mainly be due to the difference in potency on the neuromuscular transmission and not to kinetic differences.

Reversibility of kanamycin-induced neuromuscular and cardiovascular depressions by 4-aminopyridine, in comparison with neostigmine.

Antagonistic actions of 4-aminopyridine (4-AP) and neostigmine methylsulfate (NSG) on the neuromuscular and cardiovascular depressions by kanamycin (KM) were studied in urethane anesthetized rats, in comparison with D-tubocurarine (D-Tc). Measured parameters were indirectly elicited twitch of tibialis anterior muscle, mean arterial pressure and heart rate. All parameters were progressively depressed by continual infusions (0.2 ml/min) of KM (70 mg/ml) and D-Tc (10 microgram/ml). 4-AP 2.8 mg/kg s.c. significantly antagonized these depressive actions of both of D-Tc and KM, while NSG 0.1 mg/kg s.c. significantly antagonized only those of D-Tc and more strongly than 4-AP did in cardiovascular depression. 4-AP could antagonize the complete neuromuscular blockade and lethality of KM to the equilevel of conscious rats and those of D-Tc to 2.78 and 1.78 time doses of conscious rats in the complete neuromuscular blocking dose and lethal dose respectively. These results suggest that the antagonistic action of 4-AP results from its facilitation activity in the neurotransmitter release and its augmentative activity in the muscle contractility and also indicate a possibility that 4-AP can be used as a relief agent for the neuromuscular and/or cardiovascular depressions induced by some aminoglycoside antibiotics and nondepolarizing neuromuscular blocking drug in the clinical aspect.