The long latency component of retinotectal transmission: enhancement by stimulation of nucleus isthmi or tectobulbar tract and block by nicotinic cholinergic antagonists.

The optic tectum of teleosts contains high concentrations of nicotinic and muscarinic acetylcholine receptors and receives putative cholinergic innervation from both nucleus isthmi in the tegmentum and a population of intrinsic tectal cells. Using in vitro preparations of goldfish brain, we have examined the effects of cholinergic antagonists and stimulation of nucleus isthmi on the tectal response to optic nerve stimulation. Our results show that: (1) a long latency component of the retinotectal field potential is polysynaptic in origin and occurs in isolated tectum; (2) this component can spread across the tectum from a beam of stimulated fibers and can appear in areas where the monosynaptic response is small or absent; (3) both monosynaptic and long latency components of the field potential are enhanced by prior stimulation of nucleus isthmi or the tectobulbar tract (15-300 ms); (4) both the long latency component of the field potential and the effects of stimulation of nucleus isthmic or tectobulbar tract are blocked by low concentrations of nicotinic antagonists; and (5) in deeper tectum a second polysynaptic response uncovered by pharmacological block of inhibition is not blocked by nicotinic antagonists. These results indicate that the cholinergic neurons intrinsic to tectum have a role in the spread of retinotectal excitation by nicotinic actions, and that stimulation of nucleus isthmi or tectobulbar tract facilitates activity in this system. There is in addition a separate recurrent excitatory circuit in tectum.

The effects of brucine and alcuronium on the inhibition of [3H]acetylcholine release from rat striatum by muscarinic receptor agonists.

1. Radioligand binding experiments indicate that the affinity of muscarinic receptors for their agonists may be enhanced by allosteric modulators. We have now investigated if brucine can enhance the inhibitory effects of muscarinic receptor agonists on the electrically evoked release of [3H]acetylcholine ([3H]ACh) from superfused slices of rat striatum. 2. The evoked release of [3H]ACh was inhibited by all agonists tested (i.e., furmethide, oxotremorine-M, bethanechol and oxotremorine). 3. Brucine enhanced the inhibitory effects of furmethide, oxotremorine-M and bethanechol on the evoked [3H]ACh release without altering the inhibitory effect of oxotremorine. 4. Alcuronium was applied for comparison and found to diminish the inhibitory effect of furmethide on the evoked [3H]ACh release. 5. The results demonstrate that it is possible both to enhance and diminish the functional effects of muscarinic receptor agonists by allosteric modulators. 6. The direction of the observed effects of brucine and alcuronium on [3H]ACh release fully agrees with the effects of these modulators on the affinities of human M4 receptors for furmethide, oxotremorine-M, bethanechol and oxotremorine, as described by Jakubik et al. (1997). This supports the view that the presynaptic muscarinic receptors responsible for the autoinhibition of ACh release in rat striatum belong to the M4 muscarinic receptor subtype.

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.

Structural features of potent inhibitors of rat kidney histamine N-methyltransferase.

Three antimalarial drugs amodiaquine, quinacrine and chloroquine were compared side-by-side with the antiseptic agent chlorhexidine and the neuromuscular blocker alcuronium for their capacity to competitively inhibit in vitro the activity of the enzyme histamine N-methyltransferase (HMT) from rat kidney over the concentration range 10(-3)-10(-8). Amodiaquine was clearly the most potent HMT inhibitor followed by quinacrine, chlorhexidine, alcuronium and chloroquine. Investigation of the structure-activity relationships by examining space-filling models revealed marked similarities in the conformations of the arrangement of three N atoms in histamine and in each of the compounds tested.

Positive allosteric action of alcuronium on solubilized cardiac muscarinic receptors.

Alcuronium was found earlier to increase the binding of (3H)methyl-N-scopolamine [(3H)NMS] to muscarinic receptors in membranes of rat heart atria by increasing the receptors' affinity for (3H)NMS, without altering the number of (3H)NMS binding sites. We have now investigated how the interaction of alcuronium with muscarinic receptors is affected by solubilization. In experiments on pig heart atria, alcuronium had a positive allosteric effect on (3H)NMS binding both to unsolubilized receptors and to receptors solubilized by digitonin and deoxycholate. The cooperativity coefficient alpha was 0.39 +/- 0.01 for unsolubilized and 0.57 +/- 0.01 for solubilized receptors. Under the conditions used for solubilization, muscarinic receptors did not interact with G proteins, as indicated by experiments with the binding of carbachol in the absence and presence of a stable GTP analogue. Alcuronium slowed down the rates of (3H)NMS association with, and dissociation from, solubilized receptors; the dissociation rate constant was diminished more than 300 times by 30 microM alcuronium. The results show that the positive allosteric action of alcuronium on cardiac muscarinic receptors and its effect on the kinetics of radioligand binding are preserved after receptor solubilization and do not depend on the interaction of receptors with G proteins.

Antagonists discriminate muscarinic excitation and inhibition in sympathetic ganglion.

The effect of muscarinic antagonists was studied on the muscarinic slow IPSP (inhibitory postsynaptic potential) and slow EPSP (excitatory postsynaptic potential) in bullfrog sympathetic ganglia using the sucrose-gap recording method. Pirenzepine, alcuronium and atropine reduced slow IPSP amplitude more than slow EPSP amplitude. The most selective antagonists studied were pancuronium and gallamine which blocked or substantially reduced the slow IPSP without significantly affecting slow EPSP amplitude. The results suggest that the muscarinic inhibitory response may involve a different muscarinic receptor subtype, and/or receptor-ion-channel complex, than the muscarinic excitatory response.

Positive allosteric interactions on cardiac muscarinic receptors: effects of chemical modifications of disulphide and carboxyl groups.

Changes in the allosteric effects of alcuronium on rat cardiac muscarinic receptors were investigated after chemical modifications of S-S bonds or free carboxyl groups. In membranes pretreated with dithiothreitol, alcuronium lost its positive action on the binding of [3H]methyl-N-scopolamine while its inhibitory effect on radioligand dissociation was preserved. In membranes pretreated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), known to modify free carboxyl groups in proteins, the ability to bind [3H]methyl-N-scopolamine was preserved if the pretreatment had been performed in the presence of alcuronium, methyl-N-scopolamine or carbachol, while the positive cooperative effect of alcuronium on [3H]methyl-N-scopolamine binding was only preserved in membranes that had been exposed to EDC in the presence of alcuronium. Methyl-N-scopolamine, carbachol and alcuronium differed in their ability to protect (against EDC) the action of alcuronium on the rate of [3H]methyl-N-scopolamine dissociation. The results suggest that the disulphide bridge connecting the first two extracellular loops of muscarinic receptors is important for the positive allosteric action of alcuronium and that three carboxyl groups (presumably aspartate residues) are involved in receptor interactions with alcuronium and methyl-N-scopolamine. The first group is important for the effect of alcuronium on the affinity for methyl-N-scopolamine, the second is critical for the effect of alcuronium on the rate of methyl-N-scopolamine dissociation, and the third is critical for methyl-N-scopolamine binding. Presumably, the two charged nitrogens of alcuronium associate with the first and the second of the three groups involved.

Positive and negative allosteric interactions on muscarinic receptors.

It was previously found that alcuronium increases the binding of [3H]methyl-N-scopolamine to cardiac muscarinic receptors by a positive allosteric action while its effect on the binding of [3H]quinuclidinyl benzilate is negative. The, features of the antagonist's molecule which decide whether its allosteric interaction with alcuronium is positive or negative are not known. In the present work, it was found that alcuronium has a positive allosteric effect also on the binding of [3H]atropine and [3H]methyl-N-piperidinyl benzilate to muscarinic receptors in rat heart atria and that its effect on the binding of [3H]methyl-N-quinuclidinyl benzilate is negative. A comparison of the five radiolabelled antagonists that have been investigated so far indicates that the type of allosteric interaction (positive or negative) is not determined by the presence or absence of the quaternary nitrogen or of the benzilyl moiety in the molecule of the antagonist. Apparently, features of the N-bearing moiety of muscarinic antagonists other than the presence of a charge on nitrogen play a key role in the determination of the type of interaction.

Opposite effects of alcuronium on agonist and on antagonist binding to muscarinic receptors.

Alcuronium is known to retard allosterically the dissociation of [3H]N-methylscopolamine from muscarinic M2 receptors, thereby augmenting the binding of this antagonist. Functionally, alcuronium behaves as a weak antimuscarinic agent and induces in combination with N-methylscopolamine an overadditive antimuscarinic action with oxotremorine-M as the agonist. The effect of alcuronium on the binding of [3H]oxotremorine-M was studied in porcine heart homogenates. Agonist binding was concentration dependently inhibited with a Ki = 0.48 +/- 0.03 microM (means +/- S.D., n = 3). Under identical conditions [3H]N-methylscopolamine binding was elevated. Alcuronium, 100 microM, which nearly prevented the dissociation of [3H]N-methylscopolamine, retarded the rate of dissociation of [3H]oxotremorine-M only by a factor of two. These findings support the notion that the overadditive antimuscarinic action of alcuronium in conjunction with N-methylscopolamine is based on a shift by alcuronium of the interplay between agonist and antagonist in favour of the antagonist.

Potentiation by alcuronium of the antimuscarinic effect of N-methylscopolamine in guinea pig left atria.

Alcuronium is known to stabilize allosterically the binding of the muscarinic antagonist N-methylscopolamine to muscarinic M2 receptors and thus to elevate the equilibrium binding of N-methylscopolamine in homogenized cardiac tissue. In order to check for a functional consequence of this effect, the action of alcuronium alone and in combination with N-methylscopolamine was determined in contracting guinea pig left auricles with oxotremorine-M as the negative inotropic agonist. For sake of comparison, the allosteric modulator W84 = hexane-1,6-bis(dimethyl-3'- phthalimidopropyl-ammonium bromide) was included. Alcuronium displayed a weak antimuscarinic action (pA2 = 5.7). In conjunction with 10(-7) M N-methylscopolamine, alcuronium (> or = 10(-6) M) induced a more pronounced antimuscarinic effect than expected for a combination of competitive antagonists. The extent of overadditivity with combinations of W84 and 10(-7) M N-methylscopolamine was smaller. In conclusion, alcuronium potentiates the antimuscarinic effect of N-methylscopolamine in contracting cardiac preparations with high effectivity.

Positive allosteric action of eburnamonine on cardiac muscarinic acetylcholine receptors.

It was discovered recently that alcuronium and strychnine (which is a precursor of alcuronium) allosterically increase the affinity of cardiac muscarinic receptors for the antagonist, N-methylscopolamine. We have now investigated the effects of l-eburnamonine and vincamine, which are both closely related to strychnine. In experiments on rat heart atria, l-eburnamonine was found to increase the binding of [3H]N-methylscopolamine with Ehlert's cooperativity coefficient alpha = 0.35, which indicates that the strength of its allosteric action is close to that of alcuronium and strychnine (alpha = 0.31 and 0.44, respectively). However, the affinity of l-eburnamonine for the cardiac muscarinic receptors is lower than the affinities of alcuronium and strychnine (KAR = 22.6 microM, 0.15 microM, and 3.4 microM, respectively). In spite of its extremely close similarity to l-eburnamonine, vincamine has a negative allosteric effect on the binding of [3H]N-methylscopolamine (alpha = 4.1; KAR = 22.8 microM). It is likely that a systematic investigation of the allosteric effects of the analogues of strychnine will not only yield new allosteric effectors on muscarinic receptors, but also clarify the structural features responsible for the direction (positive or negative) of their allosteric effect.

Observations on the development of muscle hypersensitivity following chronic nerve conduction blockage and recovery.

Agar-sleeves containing 0.01%, 0.015% and 0.02% Tetrodotoxin were placed onto the sciatic nerve of the rat. The time-course of the conduction block and the full recovery of the nerve were studied; correlations were drawn with the hypersensitivity developed on the innervated muscles. The earliest sign of a TTX-produced conduction block was a decrease in the amplitude of the faster conducting fibres appearing 3 min later. Complete block was fully established 35 min later. The duration of a complete conduction block was a dose-dependent phenomenon and lasted from 1--4 days. The recovery process was gradual, simulating the reverse pattern of the acute TTX-block but spread over a much longer period with complete conduction recovery occuring 12 to 13 days later. Innervated muscles behaved as paralytic even before the complete establishment of a conduction block and remained so for 2--6 days after which clinical recovery was prompt. Muscles innervated by the TTX-treated nerves developed hypersensitivity to acetylcholine which could be seen within two days. This hypersensitivity continued to increase over the following days, despite some recovery of conduction. Its maximum appeared six to seven days later and then declined to return to normal at the time when nerve conduction properties had fully recovered. A similar degree of partial conduction block when acutely established always resulted in paralysis but when chronically present, the clinical picture of paralysis was fully compensated, due to the hypersensitivity of the muscle and possibly to collateral nerve sprouting.

Testing the specificity of allosteric modulators of muscarinic receptors in phylogenetically closely related histamine H1-receptors.

Gallamine, alcuronium and W84 (hexane-1,6-bis[dimethyl-3'-phthalimidopropyl-ammonium bromide]) are prototype allosteric modulators of the G-protein coupled muscarinic acetylcholine receptor family, especially of the M2-subtype. In order to probe the specificity of muscarinic allosteric modulation, we checked whether these agents interact with histamine H1-receptors which have a high homology with muscarinic receptors. Binding experiments (38 mM Na2HPO4, 12 mM KH2PO4, pH 7.5) were performed with the H1-receptor antagonist [3H]mepyramine ([3H]MEP) in guinea pig cerebellar homogenates. For the sake of comparison, binding of [3H]N-methylscopolamine ([3H]NMS) at muscarinic M2-receptors was measured in porcine cardiac homogenates under identical conditions. The modulators retarded [3H]NMS dissociation (t1/2 control=1.3 min) concentration-dependently indicating their allosteric action with half-maximum effects for gallamine at EC50,discs=27 microM, for alcuronium at EC50,diss=53 nM, and for W84 at EC50,diss=170 nM. In contrast, [3H]MEP dissociation from H1-receptors (t1/2,control=2.6 min) remained unchanged up to concentrations of 1 mM of the modulators. Equilibrium binding of [3H]NMS (KD=0.46 nM, Bmax=98 fmol/mg protein) was inhibited by gallamine, elevated by alcuronium and left almost unchanged by W84, indicating negative, positive and nearly neutral cooperativity, respectively, with the radioligand. The ternary complex model of allosteric actions yielded the equilibrium dissociation constants K(A) for the binding of the allosteric modulators to free M2-receptors: K(A,gallamine)=100 nM, K(A,alcuronium)=450 nM, K(A,W84)=69 nM. In H1-receptors, more than 1,000-fold higher concentrations than in M2-receptors were required to elicit an effect on the binding of [3H]MEP (KD=1.2 nM, Bmax=205 fmol/mg protein). Half-maximal reduction was observed at 10 mM for gallamine, 1 mM for alcuronium and 92 microM for W84. In conclusion, the muscarinic modulators have little effect on the histamine H1-receptors.

Modes of allosteric interactions with free and [3H]N-methylscopolamine-occupied muscarinic M2 receptors as deduced from buffer-dependent potency shifts.

Muscarinic M2 acetylcholine receptors contain an allosteric site that is probably located at the entrance of the ligand binding pocket above the orthosteric binding site. With the orthosteric area not occupied, allosteric agents might gain access to this site. The interaction of allosteric agents with orthoster-occupied receptors is known to depend on the buffer conditions in an alloster-specific fashion. Utilizing the buffer-dependent potency shift as an indicator, we aimed to find out for two rod-like shaped and flexible allosteric agents whether or not there is evidence for a switch in the site of attachment in free compared with [3H]N-methylscopolamine ([3H]NMS)-occupied porcine heart M2 receptors. These agents are the bispyridinium compounds WDuo3 (1,3-bis[4-(phthalimidomethoxyimino-methyl)-pyridinium-1-yl] propane dibromide) and Duo3 (4,4'-bis-[(2,6-dichloro-benzyloxy-imino)-methyl]-1,1'-propane-1,3-diyl-bis-pyridinium dibromide). The prototype allosteric agents gallamine and alcuronium were included. Inhibition of [3H]NMS association was taken to reflect alloster interaction with free receptors, inhibition of [3H]NMS dissociation indicated binding to [3H]NMS-occupied receptors. In Na,K,Pi buffer (4 mM Na2HPO4, 1 mM KH2PO4, pH 7.4 at 23 degrees C) compared with Mg,Tris,Cl,Pi buffer (45 mM Tris-HCl, 2.6 mM MgHPO4, pH 7.3 at 37 degrees C) WDuo3 underwent the same loss of potency for the interaction with either free or [3H]NMS-liganded receptors. The loss of potency was quantified by a potency ratio (PR), i.e. the ratio between the concentrations of the modulator leading to a half-maximal delay of [3H]NMS association or dissociation, respectively, in Mg,Tris,Cl,Pi compared with Na,K,Pi. For WDuo3 the ratios were PRass=27 and PRdiss=22, respectively. For Duo3, the interaction with free and [3H]NMS-occupied receptors only slightly depended on the composition of the incubation medium: PRass=1.3, PRdiss=2.8. In contrast to the other agents, the concentration-effect curves of which had slope factors nH not different from unity, the curves of Duo3 were steep (nH about -1.6). For alcuronium the shift factors amounted to PRass=29 and PRdiss=25, for gallamine to PRass=216 and PRdiss=159. In conclusion, there was a wide variation between the allosteric agents with regard to the respective buffer dependence of action. Yet, for a given allosteric agent, the interaction with either free or [3H]NMS-occupied receptors was always characterized by the same buffer-dependent shift. Thus, even the applied rod-shaped allosteric agents do not appear to switch to the orthosteric site in free compared with orthoster-occupied M2 receptors.

Interaction of the neuromuscular blocking drugs alcuronium, decamethonium, gallamine, pancuronium, ritebronium, tercuronium and d-tubocurarine with muscarinic acetylcholine receptors in the heart and ileum.

Neuromuscular blocking drugs have a high affinity for muscarinic acetylcholine receptors in the heart atria and ileal smooth muscle. In experiments on homogenates, alcuronium, gallamine, pancuronium, tercuronium and ritebronium inhibited the binding of the muscarinic antagonist (3H)quinuclidinyl benzilate (QNB) to rat heart atria with IC50 values of 0.15-0.53 mumol X 1(-1) and to ileal longitudinal muscles with IC50 values of 0.12-0.45 mumol X 1(-1). d-Tubocurarine and decamethonium inhibited (3H)QNB binding to these tissues with IC50 values of 6.2-8.5 mumol X 1(-1). For each neuromuscular blocking drug, the IC50 values were virtually identical for (3H)QNB displacement in the homogenates of the atria and of the ileal muscle. Alcuronium and gallamine differed from the other blocking agents in that they produced less steep (3H)QNB displacement curves both in the atria and the ileal muscle; Hill coefficients for the binding of alcuronium and gallamine to atrial and ileal homogenates were lower than unity. On isolated atria, gallamine, pancuronium, ritebronium and tercuronium antagonized the inhibition of tension development caused by the muscarinic agonist, methylfurmethide, with Kd values which were of the same order of magnitude as the IC50 values for the displacement of (3H)QNB binding to homogenates; the Kd of alcuronium was 12.5 times higher. d-Tubocurarine and decamethonium did not antagonize the effects of methylfurmethide at concentrations up to 100 mumol X 1(-1). On isolated ileal longitudinal muscle, gallamine and pancuronium antagonized the effects of methylfurmethide with Kd values that were 53 times and 100 times higher than their respective Kd values in the atria.(ABSTRACT TRUNCATED AT 250 WORDS)

A simple method for the quantitative evaluation of neuromuscular blockade in mice.

The purpose of this study was to develop a simple and noninvasive method for the quantitative evaluation of neuromuscular blockade in mice. The sciatic nerve in anesthetized animals (etomidate, 50 mg/kg or mephenesin, 250 mg/kg) was stimulated using transcutaneous electrodes; repetitive supramaximal stimulations (3 Hz during 3.3 sec) were applied each minute until recovery or death occurred. Evoked action potentials of the gastrocnemius muscle were recorded. The effect of each pharmacological agent was expressed as the ratio (S5/S1) of the area of the fifth response (S5) to the first one (S1). The performance of the method was tested using neuromuscular blocking agents such as alcuronium, suxamethonium, or alpha-bungarotoxin. It was concluded that the method exhibits the required statistical performances (sensitivity, repeatability, and specificity) to be recommended for in vivo investigation of neuromuscular impairment.

Alcuronium pharmacodynamics in dogs: effect-concentration relationships in the diaphragmatic and limb muscles.

Previous studies suggest that the muscles of the diaphragm are less sensitive to neuromuscular blocking agents than the limb muscles. However, this difference has not been characterized directly in terms of relaxant drug plasma concentrations. The pharmacodynamics of the non-depolarizing muscle relaxant alcuronium were therefore investigated in nine dogs using a constant-rate infusion regimen with simultaneous measurement of muscle paralysis in the limb and diaphragm. Maximum paralysis between 95 and 100% was achieved in both muscle groups, within approximately the same time interval. However, during onset of and offset of effect, the pharmacodynamic parameters ECp50 and ECp95 for the limb muscle were lower than in the diaphragm. From a pharmacodynamic effect model it was also predicted that Css(50) and Css(95) for the limb muscles are half those values for the diaphragm. Thus, the diaphragm is less sensitive to the action of alcuronium than are limb muscles. The half-time for equilibration of alcuronium between plasma and the effect site was two-fold lower for the diaphragm, and the rate of recovery from paralysis in diaphragmatic muscles was twice that observed in limb muscles. Collectively, these data suggest that there is a greater margin of safety in the diaphragmatic muscles and that the response of the peripheral limb muscles to nerve stimulation provides only a conservative index of recovery from competitive neuromuscular block in the diaphragmatic muscles.

Observations on the neuromuscular blocking action of alcuronium in the dog and its reversal by neostigmine.

The action of alcuronium chloride on neuromuscular transmission in the dogs was investigated by electrical and mechanical methods. The mean duration of action was 70 min. Reversal of its action was produced with atropine and neostigmine.

A method for recording evoked electrical and mechanical activity of muscle in the intact dog.

A technique is described for investigating the action of muscle relaxant drugs in the intact and anaesthetised dog. The ulnar nerve was stimulated at the elbow and the evoked electrical and mechanical responses were measured and recorded with an electromyograph and pen recorder.

Effect of prior administration of a non-depolarising muscle relaxant on the action of suxamethonium in the dog.

The non-depolarising muscle relaxants alcuronium (0.1 mg kg-1), gallamine (1 mg kg-1) and pancuronium (0.06 mg kg-1) were administered to six dogs. At 50 per cent return of neuromuscular activity, as measured by the train-of-four technique, the depolarising muscle relaxant suxamethonium (0.3 mg kg-1) was injected intravenously. At 50 per cent return of neuromuscular activity, atropine and neostigmine were administered to reverse the neuromuscular block. The duration of action of suxamethonium was reduced by each of the non-depolarising muscle relaxants.