Site selectivity of competitive antagonists for the mouse adult muscle nicotinic acetylcholine receptor.

The muscle-type nicotinic acetylcholine receptor has two nonidentical binding sites for ligands. The selectivity of acetylcholine and the competitive antagonists (+)-tubocurarine and metocurine for adult mouse receptors is known. Here, we examine the site selectivity for four other competitive antagonists: cisatracurium, pancuronium, vecuronium, and rocuronium. We rapidly applied acetylcholine to outside-out patches from transfected BOSC23 cells and measured macroscopic currents. We have reported the IC(50) of the antagonists individually in prior publications. Here, we determined inhibition by pairs of competitive antagonists. At least one antagonist was present at a concentration producing > or =67% receptor inhibition. Metocurine shifted the apparent IC(50) of (+)-tubocurarine in quantitative agreement with complete competitive antagonism. The same was observed for pancuronium competing with vecuronium. However, pancuronium and vecuronium each shifted the apparent IC(50) of (+)-tubocurarine less than expected for complete competition but more than expected for independent binding. The situation was similar for cisatracurium and (+)-tubocurarine or metocurine. Cisatracurium did not shift the apparent IC(50) of pancuronium or vecuronium, indicating independent binding of these two pairs. The data were fit to a two-site, two-antagonist model to determine the antagonist binding constants for each site, L(alphaepsilon) and L(alphadelta). We found L(alphaepsilon)/L(alphadelta) = 0.22 (range, 0.14-0.34), 20 (9-29), 21 (4-36), and 1.5 (0.3-2.9) for cisatracurium, pancuronium, vecuronium, and rocuronium, respectively. The wide range of L(alphaepsilon)/L(alphadelta) for some antagonists may reflect experimental uncertainties in the low affinity site, relatively poor selectivity (rocuronium), or possibly that the binding of an antagonist at one site affects the affinity of the second site.

Determination of the muscarinic receptor subtype mediating vasodilatation.

The muscarinic receptor mediating vasodilatation of the rabbit aorta and dog femoral artery has been assessed using muscarinic antagonists. With the exception of pirenzepine, the antagonist affinities were similar to those reported for the ileal receptors and dissimilar to those reported for the atrial receptors. Pirenzepine exhibited an affinity (7.54) intermediate between that reported for the CNS receptors (8.4) and that reported for the ileal receptors (6.77). This value for pirenzepine was confirmed using acetylcholine as the agonist and using the dog femoral artery as the vascular tissue. It is concluded that the muscarinic receptor profile mediating vasodilatation is not easily accommodated into the current receptor classification.

Inhibition of ionotropic neurotransmitter receptors by antagonists: strategy to estimate the association and the dissociation rate constant of antagonists with very strong affinity to the receptors.

Since binding of an agonist to an ionotropic neurotransmitter receptor causes not only channel opening, but also desensitization of the receptor, inhibition of the receptor by the antagonist sometimes becomes very complicated. The transient state kinetics of ligand association and dissociation, and desensitization of the receptor were considered on the basis of the minimal model proposed by Hess' group, and the following possibilities were proposed. 1) When an agonist is simultaneously applied to the receptor with an antagonist whose affinity to the receptor is extremely strong and different from that of the agonist, it is usually impossible to estimate the real inhibition constant exactly from the responses because desensitization of the receptor proceeds before the equilibrium of the ligand binding. Simultaneous addition of the antagonist with strong affinity to the receptor may apparently accelerate inactivation (desensitization) of the receptor. The association rate constant of the antagonist can be estimated by analyses of the rate of the inactivation in the presence and the absence of the antagonist. 2) A preincubated antagonist with a slow dissociation rate constant, i.e., a very effective inhibitor, may cause apparent noncompetitive inhibition of the receptor, since the receptor is desensitized by an agonist as soon as the antagonist dissociates from the receptor and the dissociation of the antagonist from the receptor becomes the rate-determining step. A nicotinic acetylcholine receptor (nAChR) was expressed in Xenopus oocytes by injecting mRNA prepared from Electrophorus electricus electroplax and used for the experiments on inhibition by an antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of muscarinic receptors in canine bronchial smooth muscle.

Rings of canine bronchi were suspended for isometric tension recording. Contractions produced by exogenously added acetylcholine were inhibited by pirenzepine and pancuronium. The pKB values were 6.76 for pirenzepine (calculated at 10(-6) M) and 5.30 and 5.13 for pancuronium (calculated at 10(-5) and 3 X 10(-5) M, respectively). Contractile responses evoked by cholinergic nerve stimulation (0.2-16 Hz, 9 V) were depressed by pancuronium in a concentration-dependent manner, while concentrations of pirenzepine selective for M1-muscarinic receptors were without effect. The results indicate that exogenous and nerve-released acetylcholine activate a homogenous population of M2-muscarinic receptors in isolated preparations of canine bronchial smooth muscle.

Isobolographic analysis of non-depolarising muscle relaxant interactions at their receptor site.

Administration of certain combinations of non-depolarising muscle relaxants produces greater than expected neuromuscular blockade. Synergistic effects may be explained by drug interactions with the postsynaptic muscle nicotinic acetylcholine receptor. To investigate this hypothesis, the adult mouse muscle nicotinic acetylcholine receptor (alpha(2)beta delta epsilon) was heterologously expressed in Xenopus laevis oocytes and activated by the application of acetylcholine (10 microM). The effects of five individually applied muscle relaxants and six combinations of structurally similar and dissimilar compounds were studied. Drug combinations containing equipotent concentrations of two agents were tested and dose-response curves were determined. All compounds tested alone and in combination produced rapid and readily reversible, concentration-dependent inhibition. Isobolographic and fractional analyses indicated additive interactions for all six tested combinations. These findings suggest that synergistic neuromuscular blocking effects, observed for the administration of certain combinations of muscle relaxants, do not result from purely postsynaptic binding events at the muscle nicotinic acetylcholine receptor, but rather from differential actions on pre- and postsynaptic sites.

Open channel and competitive block of nicotinic receptors by pancuronium and atracurium.

Mouse myotubes were used to investigate effects of the nondepolarizing neuromuscular blocking drugs pancuronium and atracurium on embryonic-type nicotinic acetylcholine receptor channels. Experiments were performed using patch-clamp techniques in combination with devices for ultra-fast solution exchange at outside--out patches. Application of 0.1 mM acetylcholine resulted in a fast current transient. When the peak amplitude was achieved, the current decayed monoexponentially due to desensitization. After application of drugs (pancuronium or atracurium), two different mechanisms of block were observed: (1) open channel block of embryonic-type nicotinic acetylcholine receptor channels after coapplication of blocker and acetylcholine, characterized by decrease of the time constant of current decay; (2) competitive block of embryonic-type nicotinic acetylcholine receptor channels by pancuronium or atracurium after preincubation of outside-out patches with the respective blocker. Different affinities of pancuronium (K(B) approximately 0.01 microM) and atracurium (K(B) approximately 1 microM) to embryonic-type nicotinic acetylcholine receptor channels were observed.

Pancuronium and gallamine are antagonists for pre- and post-junctional muscarinic receptors in the guinea-pig lung.

The effects of atropine, pancuronium and gallamine were tested on pre- and post-junctional muscarinic receptors in the lung. Inhibition of bronchoconstriction induced by intravenous injection of acetylcholine (ACh) was used as a measure of post-junctional receptor blockade. All three antagonists reduced ACh-induced bronchoconstriction. The effects were dose-related for atropine and pancuronium and complete inhibition was obtained with 0.01 mg/kg and 10 mg/kg respectively. Gallamine was much less potent than the other two drugs; the inhibitory effect was not dose-related and never exceeded 50% even at a dose of 10 mg/kg. In contrast, blockade of pre-junctional inhibitory muscarinic receptors in pulmonary parasympathetic nerves by these three antagonists, produced potentiation of bronchoconstriction induced by vagal-nerve stimulation. Consequently, the effect of the three antagonists on vagally-induced bronchoconstriction is dependent on the balance between their pre- and post-junctional blocking activity. Gallamine was the most effective and atropine the least effective antagonist for potentiating nerve-induced bronchoconstriction. At doses which produce 100% neuromuscular blockade, both pancuronium (0.04 mg/kg) and gallamine (4 mg/kg) potentiated vagally-induced bronchoconstriction. At these doses, pancuronium doubled and gallamine caused a four-fold increase in vagally-induced bronchoconstriction, despite partial concurrent blockade of muscarinic receptors in the smooth muscle of the airways.

Differential effects of pancuronium bromide on cardiopulmonary function in the neonatal lamb.

The effect of pancuronium bromide (Panc Br) on resting cardiopulmonary function and cardiopulmonary responses to intravenous injection of acetylcholine (Ach) and histamine (H) was evaluated in neonatal lambs. The animals were mechanically ventilated and managed to maintain physiologic gas exchange and acid-base conditions. A proximal segment of the cervical trachea was bypassed; the developed pressure response of this segment (P cervical trach) was used as a direct indication of airway smooth muscle contraction and bronchoconstriction. Pulmonary resistance (Rp) and functional residual capacity were determined. The change in Rp from resting values was used as a functional indicator of central and peripheral airway bronchoconstriction. Cardiovascular function and responses were evaluated from changes in mean arterial pressure and heart rate. Following Panc Br, there was a significant reduction in Ach-induced P cervical trach (-50 +/- 9.2% SE) and Rp (-46 +/- 2.4% SE). In contrast, Panc Br did not significantly change Ach-induced bradycardia and hypotension, cardiopulmonary responses to H, and resting cardiopulmonary function. The differential effects of Panc Br on cardiopulmonary function appear to be related to regional differences between cardiovascular and airway smooth muscle muscarinic receptors in the neonate. The results of this study elucidate a mechanism which may explain previously reported variability in the effect of Panc Br on neonatal cardiopulmonary function. Furthermore, the Panc Br-related attenuation of airway smooth muscle responses suggests that this form of neuromuscular blockade affects the regulation of airway tone and may influence the susceptibility of the neonate to airway deformation consequent to mechanical ventilation.

Cardiac vagolytic action of some neuromuscular blockers.

Cardiac vagolytic effect of four commonly used neuromuscular blockers, (viz. D-tubocurarine, decamethonium, pancuronium and gallamine) was compared in midcollicular decerebrate rats. The intravenous doses of neuromuscular blockers used (d-tubocurarine: 0.1 mg/kg; decamethonium: 2 mg/kg; pancuronium: 0.1 mg/kg; gallamine: 20 mg/kg) were sufficient to produce the paralysis of respiratory muscles. Bradycardia was induced by electrical stimulation of the vagus or by injecting dimethyl-phenyl-piperazinium (DMPP; a ganglionic stimulant). It was observed that d-tubocurarine and decamethonium were devoid of cardiac vagolytic action. On the other hand, pancuronium and gallamine inhibited significantly the bradycardia induced by electrical stimulation of the vagus or injection of DMPP; gallamine was found to have greater vagolytic action. The pressor responses to DMPP were not attenuated by pancuronium and gallamine indicating that in the dose administered, these agents did not block the ganglia. Bradycardia induced by the administration of acetylcholine in the left atrium was also attenuated by pancuronium and gallamine suggesting that the drugs produce cardiac vagolytic action by acting on the post-synaptic cholinergic receptors of the heart.

On mechanism of action of ethanol-induced contraction of frog rectus abdominis.

Ethanol in low doses (0.5 to 4 M) causes contraction of isolated frog rectus abdominis muscle. Higher concentration did not produce any further increase in maximum response. Pretreatment with dantrolene produced partial but equal inhibition of acetylcholine (Ach) induced as well as ethanol-induced contraction in equieffective doses. Pretreatment with pancuronium produced right and downward shift of ethanol induced contraction. Pretreatment with succinylcholine produced persistent contraction of tissue and this response remained unaffected on subsequent treatment with Ach as well as ethanol. Pretreatment with hemicholinium abolished ethanol induced contraction, although tissue remained viable as confirmed on addition of Ach. The contraction induced by ethanol decreased on pretreatment with dantrolene as well as in Ca2+ free ringer. The results indicate that ethanol induced contraction may be due to release of Ach or Ach like neurotransmitter at neuromuscular junction and calcium acts as mediator to produce these effects.

Cholinergic mechanisms and bronchoconstriction in guinea-pigs.

Seventy-four guinea-pigs have been actively sensitized against ovalbumin. The effects of bilateral cervical vagotomy and of cholinergic efferent blockade on the bronchoconstriction induced by inhaled acetylcholine, histamine or antigen have been quantified. Animals were curarized then tested during controlled ventilation to ensure constant conditions throughout the whole experiment by suppressing the post-vagotomy apneusis. Bronchial sensitivity was estimated by calculating the bronchoconstrictor dose-threshold provoking respiratory asynchronism. Corresponding variation of total lung resistance and dynamic compliance were also measured as indices of bronchial reactivity. The data obtained in 27 guinea-pigs indicated that the vagi section produced no significant effect on the direct bronchial response to histamine (P greater than 0.5), to acetylcholine (P greater than 0.1) or to antigen (P greater than 0.5). In 47 guinea-pigs, atropine at doses 5 and 10 times higher than the acetylcholine blocking one did not alter the direct bronchial response to histamine (P greater than 0.5). Higher doses induced a progressive decrease in bronchial sensitivity to antigen (.05 greater than P greater than .01) which could be related to the repetitive administration of the antigen. According to the present results, peripheral cholinergic mechanisms play no significant role in guinea-pigs neither in histamine nor in anaphylactic bronchoconstriction. Curarization has no influence on resting bronchial tone.

The myocardial effects of pancuronium.

The effect of pancuronium on myocardial contractility was studied in three different animal preparations. Pancuronium produced no change in isometric contraction of rabbit atrial or cat papillary muscle but displaced the acetylcholine dose-response curve to the right in the papillary muscle preparations, verifying a muscarinic blocking effect of this drug. In atropinized dogs in vivo pancuronium produced no significant change in the cardiovascular parameters studied. These studies show that pancuronium exerts its cardiovascular effects primarily by blocking muscarinic receptors in the heart.

Interactions between calcium entry blockers and vecuronium bromide in anaesthetized cats.

Possible interactions between three calcium entry blocking agents (nifedipine, verapamil and bepridil) and the non-depolarizing neuromuscular blocking agent, vecuronium bromide, were investigated in chloralose-anaesthetized cats. In i.v. doses which caused decreases in arterial pressure, the calcium entry blockers did not affect indirectly-elicited twitches of tibialis muscle, but did potentiate the effects of vecuronium. No such potentiation of vecuronium was observed with the vasodilator drug, sodium nitroprusside. Experiments using close-arterial injections of acetylcholine suggested that the probable site of interaction is on the postsynaptic muscle membrane and probably involves blockade of calcium channels in skeletal muscle.

In vitro study of interactions between i.v. anaesthetics and neuromuscular blocking agents.

The interactions of four anaesthetic drugs (ketamine, propanidid, Althesin and methohexitone) with two neuromuscular blocking agents (suxamethonium and pancuronium) have been investigated. On the isolated rat phrenic nerve-diaphragm preparation, all the anaesthetic drugs examined potentiated suxamethonium more than they potentiated pancuronium. The anticholinesterase agent, ecothiopate, had no significant effect on the potentiation of suxamethonium caused by the anaesthetic drugs. At low concentrations the anaesthetic drugs increased the twitch tension elicited by stimulation of the phrenic nerve, whilst at high concentrations this potentiation was followed by blockade of neuromuscular transmission. With the exception of Althesin, all the anaesthetics decreased the sensitivity of the frog rectus preparation to acetylcholine. The possible sites and mechanisms of these interactions are discussed.

Effect of drugs, hormones and electrical field stimulation on isolated muscle strips from human choledochoduodenal junction.

The behaviour of in vitro strips from the human choledochoduodenal junction would appear to be related to the anatomical location of origin of the strip. Strips from the papillary region showed low tone and obvious spontaneous rhythmic contractions (0 X 5-6/min). Strips from the region of the inferior choledochal sphincter showed, in ten out of fifteen specimens, spontaneous myogenic tone and gave a relaxation or a biphasic response (relaxation followed by contraction) to electrical field stimulation (0 X 3 ms pulses at 10 Hz for 5 s). All strips from human choledochoduodenal junction are remarkably insensitive to a variety of gastrointestinal hormones and to opioid agents.

Effect of bufalin and pregnanes on vasoreactivity of human resistance arteries.

Endogenous Na/K ATPase inhibitory activity has been implicated in salt and water homeostasis in mammals and amphibians. Recent interest has focused on endogenous cardiac glycosides, some progesterone derivatives (pregnanes) and the amphibian bufodienolides. This study has examined the effects of non-planar and planar pregnanes and the bufodienolide bufalin on vasoreactivity of human resistance arteries. Bufalin and a non-planar pregnane caused concentration-dependent potentiation of the tone of submaximally pre-contracted arteries and inhibited endothelium-dependent relaxation, whereas a planar pregnane affected neither response. The relative potency of the compounds studied suggest the results do not simply reflect degrees of Na/K ATPase inhibition. The active compounds may be important in the regulation of vascular tone.

Activation of brain acetylcholine receptors by neuromuscular blocking drugs. A possible mechanism of neurotoxicity.

BACKGROUND: Neuromuscular blocking drugs cause excitement and seizures when introduced into the central nervous system. We examined the possibility that these drugs produce paradoxical activation of acetylcholine or glutamate receptors, the chief types of brain receptors involved in excitatory neurotransmission. METHODS: Because activation of central glutamate or acetylcholine receptors causes calcium influx into postsynaptic neurons, we measured intracellular calcium concentration ([Ca2+]i) as an index of receptor activation. Changes in [Ca2+]i were compared in brain slices exposed to neuromuscular blocking drugs or acetylcholine and glutamate receptor agonists. [Ca2+]i was measured with the fluorescent dye fura-2. RESULTS: Pancuronium and vecuronium caused sustained increases in [Ca2+]i in approximately the same potency ratio as for seizure activity in vivo (concentrations at which the increase in [Ca2+]i was 95% of maximal: 100 and 400 microM, respectively). Atracurium and laudanosine did not increase [Ca2+]i in cortical slices. Increases in [Ca2+]i caused by both pancuronium and vecuronium were prevented by the non-subtype-specific nicotinic acetylcholine receptor antagonist D-tubocurarine and were reduced 44-73% by atropine. Blockade of glutamate receptors or voltage-gated calcium or sodium channels had no effect on calcium influx. CONCLUSIONS: The results suggest that the acute excitement and seizures caused by introduction of pancuronium and vecuronium into the central nervous system is due to accumulation of cytosolic calcium caused by sustained activation of acetylcholine receptor ion channels.