The effects of acute and chronic hydrocortisone treatment on neuromuscular blockade in the anesthetized cat.

It is fairly widespread clinical practice to administer large doses of corticosteroids to patients in cases of shock; doses of hydrocortisone as high as 50 mg X kg-1 given intravenously have been proposed and used. Hydrocortisone, when administered in this way during surgery, has been implicated in interactions with neuromuscular blocking agents. In order to determine the type and mechanism of this interaction, the authors undertook further investigation. The effects of hydrocortisone were studied in two ways. Firstly, a constant 50% depression of the indirectly elicited twitch tension of the tibialis-anterior muscle was established in cats, using a constant intravenous infusion of either pancuronium (1.0 +/- 0.2 micrograms X kg-1 X min-1) or succinylcholine (3.6 +/- 0.8 micrograms X kg-1 X min-1). The effects of intravenous hydrocortisone then were studied on this block. Secondly, cats chronically were treated with 2 mg X kg-1 of intramuscular hydrocortisone three times a week for 1 month, and then dose-response curves were constructed for pancuronium or succinylcholine. Acute administration of intravenous hydrocortisone (1-15 mg X kg-1) alone had no affect on the twitch tension of either the tibialis-anterior or soleus muscles, however, the corticosteroid (7 and 15 mg X kg-1) did significantly (P less than 0.05) enhance the 50% depression of the indirectly elicited twitch tension of the tibialis-anterior muscle produced by the constant intravenous infusion of pancuronium. The soleus muscle was affected similarly (n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)

Potentiation of neuromuscular blockade by verapamil.

The effects of intravenous (iv) verapamil (0.01 to 1.0 mg/kg) on the constant neuromuscular block produced by an iv infusion of either pancuronium or succinylcholine were studied on the indirectly stimulated gastrocnemius and tibialis-anterior muscles of the rabbit anesthetized with halothane in oxygen. Verapamil alone (n = 6) had no significant effect. However, the drug did significantly potentiate the 50% twitch depression of the gastrocnemius muscle produced by a constant iv infusion of either pancuronium (n = 5) or succinylcholine (n = 5) to 36 +/- 6% and 45 +/- 1% of control, respectively. This effect of verapamil occurred with doses of 0.1 mg/kg for pancuronium and 0.01 mg/kg for succinylcholine; these doses of verapamil were the lowest which produced a significant effect. In contrast, verapamil had no significant effect on the progression of the neuromuscular blockade of either the gastrocnemius or tibialis-anterior muscles produced by alpha-bungarotoxin (n = 5). Verapamil also significantly prolonged the P-R interval of the ECG from a control value of 71 +/- 2 ms to 78 +/- 3 ms at a dose of 0.1 mg/kg and to 93 +/- 6 ms at a dose of 0.3 mg/kg. The possible mechanisms of the neuromuscular actions of verapamil are discussed and it is concluded that verapamil can produce potentiation of either pancuronium- or succinylcholine-induced neuromuscular block at doses within the therapeutic range.

Org. 6368: a nondepolarizing neuromuscular blocking agent with a novel effect on end-plate conductance.

The effects of the nondepolarizing agent pancuronium and three derivatives on end-plate currents (e.p.c.s), evoked by neural stimulation at the amphibian neuromuscular junction, were investigated using conventional voltage clamp techniques. All four agents depressed peak e.p.c. amplitude and also shortened the exponential time constant of the e.p.c. decay compared with control. The properties of one derivative, Org. 6368, were investigated in detail and revealed the drug to produce marked blockade of end-plate ionic conductance relative to receptor blockade. Analysis of the kinetic behavior of Org. 6368 revealed a novel mechanism of channel blockade and dissociation which is very different to that observed with some of the local anesthetics. The rate of association of Org. 6368 with the channel binding site, 1.92 X 10(7) M-1 sec-1 at 0 mV, had virtually no dependence on membrane voltage with an e-fold increase with a change of voltage of 2940 mV while the rate of dissociation, 26.4 sec-1 at 0 mV, exhibited a marked dependence on voltage with an e-fold increase with a change of voltage of only -39.5 mV. It is proposed that the rate of association of Org. 6368 is rate limited by diffusion while the rate of dissociation from the channel binding site is increased by hyperpolarization, due to the drug molecule being attracted into the membrane field before leaving the binding site.

Neuromuscular and vagal blocking actions of pancuronium bromide, its metabolites, and vecuronium bromide (Org NC45) and its potential metabolites in the anaesthetized cat.

The neuromuscular and cardiac vagus blocking actions of pancuronium, vecuronium (Org NC45) and their respective potential hydroxy metabolites have been studied in the chloralose-anaesthetized cat. Pancuronium was three times more potent as a neuromuscular blocker than its 3-hydroxy derivative, 20 times more potent than the 17-hydroxy derivative and 45 times more potent than the 3,17-dihydroxy derivative. The vagal:neuromuscular block ratios measured at 50% inhibition for these compounds were pancuronium 3.0, 3-hydroxy derivative 6.4, 17-hydroxy derivative 1.1 and 3,17-dihydroxy derivative 0.36 (a value greater than unity indicated greater potency at the neuromuscular junction). Vecuronium was 1.4 times more potent than its 3-hydroxy derivative, 24 times more potent than the 17-hydroxy derivative and 72 times more potent than the 3,17-dihydroxy derivative as a neuromuscular blocker. The vagal:neuromuscular block ratios were vecuronium 79.8, 3-hydroxy derivative 40.4, 17-hydroxy derivative 0.85 and 3,17-dihydroxy derivative 0.15. The 3-hydroxy derivative of vecuronium, the most likely first metabolite of vecuronium, thus possessed only slightly less neuromuscular blocking potency than vecuronium, coupled with a high safety margin between neuromuscular and vagal blocking doses. In addition, the time-course of its action was not different from that of vecuronium. Thus, it is concluded that this potential metabolite is unlikely to give rise to tachycardia in man. It is unlikely that the 17-hydroxy and 3,17-dihydroxy derivatives of vecuronium would be produced in sufficiently great quantities by metabolism from vecuronium to result in either tachycardia or residual neuromuscular blockade.

A comparison of 3,4-diaminopyridine and 4-aminopyridine in the anaesthetized cat.

3,4-Diaminopyridine and 4-aminopyridine were compared in the anaesthetized cat and found to be equiactive in their anti-curare activity. This in vivo similarity is at variance with previous in vitro studies which demonstrate 3,4-diaminopyridine to be more active than 4-aminopyridine at the neuromuscular junction. Possible reasons for the similarity between the two aminopyridines at the in vivo neuromuscular junction are discussed and it is concluded that 3,4-diaminopyridine has only marginal advantages over 4-aminopyridine as a potential anti-curare agent.

Cumulation of neomycin and its residual potentiation of tubocurarine in the cat.

Cumulation and interaction of neuromuscular blocking effects of neomycin and tubocurarine were determined on responses of the tibialis anterior muscle of the anaesthetized cat to indirect stimulation. Neuromuscular block produced by neomycin was cumulative despite rapid and apparent full recovery of twitch tension and an interval of 3 h between dose-response studies. Ed50 of neomycin on twitch tension decreased (P less than 0.05) from 22 +/- 4 mg kg-1 to 12 +/- 3 mg kg-1 after 3 h; marked post-tetanic exhaustion was observed after the antibiotic. Neuromuscular block (80-90%) with neomycin 25-30 mg kg-1 followed by full recovery and 3-h interval decreased (P less than 0.05) the ED50 of tubocurarine on twitch tension from 0.2 +/- 0.02 mg kg-1 to 0.13 +/- 0.03 mg kg-1. Tubocurarine exhibited no cumulative effects at intervals of 2 h and did not significantly affect the response of twitch and tetanic tension to neomycin after an interval of 2 h. It is concluded that neomycin increases the sensitivity of neuromuscular transmission to tubocurarine despite apparently normal responses to indirect stimulation and that the post-tetanic exhaustion observed with neomycin alone may explain apnoea reported in patients with the antibiotic.

Characterization of end-plate conductance in transected frog muscle: modification by drugs.

Cutaneous pectoris muscles of Rana pipiens were transected distal to the innervated region. Within 10 min, membrane potentials (Em's) of -33 +/- 2.5 mV and end-plate potentials (3-15 mV) were recorded unaccompanied by muscle action potentials or twitch. The fall in Em was associated with a net loss of [K+]i and a net gain of [Na+]i. Although input resistance fell by 50% and the space constant was slightly reduced in the transected muscle fibers, end-plates could be adequately voltage-clamped with two microelectrodes. End-plate currents (e.p.c.s) with rise times of 350 to 700 musec were recorded as a function of holding potential (Vm). The current-voltage relationship of peak e.p.c.s over the range of -70 to +20 mV was linear and the reversal potential (-6.6 +/- 2.2 mV) was the same as that found for intact muscle fibers. The decay phase of e.p.c.s could be described as a single exponential at all Vm's and had a voltage and temperature dependence similar to that described for e.p.c.s of glycerol-treated muscles. Tubocurarine (0.3 microM) caused a significant decrease in the time constant (tau) of e.p.c. decay and e.p.c. amplitude. The depression of e.p.c. amplitude by tubocurarine was reversed by 4-aminopyridine while the decrease of tau was not. Atropine (10(-4) M) caused a monotonic shortening of e.p.c.s at a Vm of -90 mV but e.p.c.s recorded at +50 mV were biphasic. Lidocaine, a quaternary nitrogen analog of lidocaine (QX314), lobeline and hexafluorenium were studied also in transected muscle and their effects on the parameters of e.p.c. are described. Both lobeline (50 microM) and hexafluorenium caused a decrease of tau and eliminated the voltage dependence of tau at negative Vm's. The transected muscle can be used for the study of conductance kinetics of end-plate and for the study of drug action uncomplicated by the presence of other drugs of Mg++ to eliminate contraction.

The action of polymyxin B at the frog neuromuscular junction.

1 The effects of polymyxin B at the neuromuscular junction of the frog were studied by conventional electrophysiological and voltage clamp techniques. 2 At a concentration of 2.5 micrograms/ml polymyxin B produced neuromuscular blockade in 10 to 15 min neuromuscular block was characterized by a depressed e.p.p. quantal content (28 plus or minus 7), which was similar to that determined from endplates exposed to 13 mM magnesium (23 plus or minus 3), and a low e.p.p. quantal size, which was similar to that determined from endplates exposed to 3 microM (+)-tubocurarine. 3 Polymyxin B (0.25 to 0.75 micrograms/ml) decreased mean miniature e.p.pl amplitude with little effect on frequency. At a concentration of 5 micrograms/ml polymyxin B markedly shortened the duration of endplate currents (e.p.cs) and abolished the relationship between holding potential and the time to half-decay at negative potentials greater than -60 mV. This action is consistent with block of open acetylcholine activated ionic channels. 5 4-Aminopyridine (20 micrometers) antagonized the depressed e.p.p. quantal content produced by polymyxin B but did not alter the shortened e.p.c. duration. 6 It is concluded that polymyxin B decreases quantal release and produces some degree of postjunctional receptor blockade and a marked and persistent blockade of of acetylcholine activated channels. The latter action may explain the difficulty of reversal of polymyxin B-induced neuromuscular blockade and its non-competitive nature.

Reversal of dantrolene sodium-induced depression of skeletal muscle in the cat.

Dantrolene sodium, a muscle relaxant, does not have a clinically useful antagonist. The present study was undertaken to test the efficacies of germine monoacetate, 4-aminopyridine, neostigmine, and calcium chloride as possible agents for the reversal of the direct skeletal muscle depression produced by dantrolene sodium in the cat anesthetized with alpha-chloralose. Depression of the indirectly elicited twitch responses (0.1 Hz) of the tibialis anterior muscle by 25, 50, 75 and 84 per cent was produced by dantrolene, 0.16, 0.36, 0.88 and 1.13 mg/kg respectively; spontaneous recovery of twitch tension during the subsequent 30 min was negligible. After the 30-min observation period had elapsed, one of the reversal agents under study was given (iv) in divided doses at intervals of 10 min (five cats for each agent). Germine monoacetate (2 X 0.5 mg/kg) immediately reversed the dantrolene-induced twitch depression, with an over-shoot that persisted for an hour. 4-Aminopyridine (4 X 0.5 mg/kg) caused a steady but incomplete reversal to 17 per cent depression, 30 min after the last dose. Neostigmine (4 X 0.04 mg/kg) caused an immediate, but transient, reversal of the twitch depression, with overshoot. Calcium chloride (4 X 10 mg/kg) was without effect. It is concluded that germine monoacetate is the drug of choice for reversal of the muscle depression produced by dantrolene sodium in the cat.

The action of dantrolene on transmitter mobilization at the rat neuromuscular junction.

Dantrolene sodium (20 microM) was found to decrease transmitter mobilization and the apparent available store of acetylcholine at frequencies of nerve stimulation of 50 and 100 Hz at the neuromuscular junction of the rat hemidiaphragm preparation treated with 2 microM (+)-tubocurarine. This effect of dantrolene sodium was not as marked at frequencies of nerve stimulation of 25 Hz or less, also no significant effect of the drug was observed on the amplitude of endplate potentials (EPPS) at any frequency of nerve stimulation. No effect of dantrolene sodium (20 microM) on mean miniature EPP amplitude or frequency was observed. It is suggested that the action of dantrolene sodium on mean EPP quantal content may be due to an action on stores of bound calcium within the motor nerve terminal. This effect of the drug is unlikely to be of physiological consequence in vivo, since it was only observed at high frequencies of nerve stimulation and did not cause a significant reduction of EPP amplitude during trains of stimuli.

The effects of 3,4-diaminopyridine on acetylcholine release at the frog neuromuscular junction.

The effects of 3,4-diaminopyridine (3,4-DAP) were studied on spontaneous and evoked transmitter release in the frog sartorius muscle preparation. At a concentration of 1 mM, 3,4-DAP caused the appearance of spontaneous giant potentials, this effect of 3,4-DAP also occurred in absence of extracellular calcium. At lower concentrations in magnesium Ringer 3,4-DAP (1-10 microM) increased e.p.p. quantal content without causing the appearance of spontaneous giant potentials, 3,4-DAP prolonged muscle action potential (1 mM) and it is suggested that prolongation of the nerve terminal action potential by 3,4-DAP contributes largely to the increased evoked acetylcholine release. The ability of 3,4-DAP to cause the appearance of giant spontaneous potentials is considered to be independent of the drug's known ability to inhibit potassium conductance in excitable membranes, and may be due to the release of performed multiquantal packets of acetylcholine as suggested for vinblastine. It is concluded that 3,4-DAP is a useful tool in the study of both evoked and spontaneous acetylcholine release.

4-Aminopyridine reversal of sympathetic ganglionic blockade in the anesthetized cat.

The effects of 4-aminopyridine on the blockades of transmission of the superior cervical ganglion produced by hexamethonium, d-tubocurarine, and polymyxin B were investigated in 15 anesthetized cats. Isometric contractions of the nictitating membrane resulting from pre- and postganglionic stimulation were quantified. A 75-85 per cent ganglionic blockade produced by hexamethonium, 3 mg/kg, or by d-tubocurarine, 1.0 to 1.2 mg/kg, was completely reversed by 4-aminopyridine, 1 mg/kg. After injection of 4-aminopyridine, the times required for the contractions of the nictitating membrane to increase from 25 to 75 per cent of control with preganglionic stimulation were 6 +/- 3 (mean +/- SEM) and 7 +/- 2 min for hexamethonium and d-tubocurarine, respectively. Both values are significantly (P less than 0.05) shorter than their respective control spontaneous recovery times of 26 +/- 8 and 25 +/- 7 min. Polymyxin B, 20-35 mg/kg, depressed the contractions of the nictitating membrane resulting from pre- and postganglionic stimulation by 84 +/- 2 and 66 +/- 10 per cent, respectively. 4-Aminopyridine, 1 mg/kg, lessened the polymyxin B-induced depression of the contractions of the nictitating membrane resulting from preganglionic stimulation to 50 +/- 13 per cent, but had less effect, 61 +/- 13 per cent, on the contractions resulting from postganglionic stimulation. It is concluded that 4-aminopyridine rapidly and completely reverses the sympathetic ganglion blackade produced by hexamethonium or d-tubocurarine, and is partially effective in the reversal of the autonomic effects of polymyxin B.

Pharmacology of ORG NC 45 compared with other non-depolarizing neuromuscular blocking drugs.

From results of pharmacological tests on the neuromuscular and autonomic blocking actions of a series of pancuronium analogues, Org NC 45, the C16 monoquaternary analogue of pancuronium, was selected for detailed study. Org NC 45 has a non-depolarizing mechanism of action, is more rapid in onset and shorter in duration of action than pancuronium. It shows less cumulation than pancuronium or tubocurarine, and is easily antagonized by anticholinesterases and aminopyridines. Org NC 45 exhibits a low propensity to release histamine. Its ability to inhibit cholinesterases is not likely to be important at neuromuscular blocking doses. Org NC 45 possesses negligible ganglion-blocking activity and there is a wide margin between neuromuscular and vagal blocking doses. Thus cardiovascular side-effects are unlikely to occur with the use of Org NC 45. It will hydrolyse mainly to its 3-hydroxy analogue which, like Org NC 45, possesses a wide margin between neuromuscular and vagal blocking doses. Org NC 45 has a high selectivity for the neuromuscular junction and represents a potentially useful addition to the armamentarium of clinically useful muscle relaxants.

The neuromuscular and autonomic blocking activities of pancuronium, Org NC 45, and other pancuronium analogues, in the cat.

Twenty-six mono- or bis-quaternary salts of 3,17-dioxy-2 beta, 16 beta-dipiperidino-5 alpha-androstanes (including pancuronium) and one 17-desoxy congener were tested for neuromuscular blocking and autonomic blocking activities in the chloralose-anaesthetized cat. The 17 beta-acetoxy series, all the members of which contain an acetylcholine-like fragment in the steroidal D-ring, was most selective for effecting neuromuscular blockade. The salient member of this series is 3 alpha, 17 beta-diacetoxy-2 beta, 16 beta-dipiperidino-5 alpha-androstane 16 beta-N-monomethobromide (Org NC 45) which is highly selective in blocking neuromuscular transmission in that a dose approximately sixty times greater than the neuromuscular blocking dose was required to block responses to vagal stimulation. In contrast, in doses sufficient to produce neuromuscular block, pancuronium simultaneously blocked responses to vagal stimulation. Moreover, pancuronium and Org NC 45 exhibited the same order of neuromuscular blocking activity and therefore the latter potentially represents a useful addition to the armamentarium of neuromuscular blocking agents currently in clinical use.

Inhibition of aminopyridine-induced contractile activity in skeletal muscle by tetrodotoxin and by magnesium.

The effects of tetrodotoxin and magnesium have been studied on aminopyridine-induced contractile activity seen in the absence of nerve stimulation. In the chick biventer cervicis muscle both tetrodotoxin and magnesium pretreatment prevented the development of fasciculations and contractures in the presence of 4-aminopyridine and 3,4-diaminopyridine. Both tetrodotoxin and magnesium abolished aminopyridine induced fasciculations and contractures. Tetrodotoxin did not reduce postjunctional sensitivity to the agonists acetylcholine and carbachol whereas magnesium produced some reduction of postjunctional sensitivity. It is concluded that conducted action potentials must be involved in the aminopyridine-induced contractile activity. In the frog sartorius muscle aminopyridines occasionally induced repetitive endplate potentials suggesting that the compounds induce repetitive nerve activity. In both tetrodotoxin and magnesium treated preparations 4-aminopyridine produced only a moderate increase in miniature endplate potential frequency. It is concluded that aminopyridines increase nerve membrane excitability resulting in the generation of repetitive action potentials in the absence of nerve stimulation.